METHOD AND COMPOSITION FOR NUCLEIC ACID STORAGE FROM

BLOOD FRACTIONS

FIELD

[0001] The present application pertains to the field of nucleic acid technology. More particularly, the present application relates to a method and aqueous composition for the collection, stabilization, transport, storage, and banking/ar chiving of nucleic acid, particularly DNA, in whole blood, plasma, buffy coat, and white blood cell fractions at ambient temperature for extended periods of time, which can then be isolated and/or analyzed using conventional molecular biology methods.

BACKGROUND

[0002] The preservation and storage of nucleic acid is of interest to scientists in a wide range of fields and disciplines. In medical genetics, research projects frequently require the accumulation of large numbers of biological samples or biospecimens over an extended period of time, with analysis typically performed at the end of the gathering period, when a series is complete. An area of concem, when considering results obtained from such research projects is the possibility of nucleic acid degradation in storage and other events or factors which could impact the quality of the genetic material. The same risks apply to the storage of biospecimens for diagnostic purposes, with chemical degradation being the major threat to nucleic acid preservation. [0003] A Biobank or Biorepository collects biological materials or specimens and related data often from tens or hundreds of thousands of individuals, processes or prepares samples for long-term storage, stores or archives unprocessed and or processed biological materials, and manages the distribution/disposition of biospecimens to support future scientific investigation of, for example, genetic disorders, drug discovery and development, as well as personalized genomic medicine. Although Biorepositories have existed in some form for over 60 years, their recent surge in numbers, size, and prominence coincides with success in sequencing the human genome in 2003, the subsequent explosion of new bioinformatics technologies and the development of next-generation sequencing. Along with these new technologies comes the vision of improved health through genomic medicine. Sequencing of the human genome has greatly facilitated the shift towards studying multi-factorial disorders, rather than rare monogenic disease. These studies, however, are highly dependent on the ability to establish clear disease-genomic profile associations which requires large epidemiological studies and the availability of biological samples from well-characterized patient cohorts.

[0004] A critical feature of any nucleic acid-based assay or genetic diagnostic test is the quality of the up-front sample preparation methods and stabilizing conditions for collected biospecimens. Target nucleic acid must be of sufficient quality and quantity for subsequent processes such as amplification or hybridization, even following years of storage in archived biospecimens, such as frozen whole blood or frozen buffy coat. Typically, following the collection of a whole blood sample into a tube containing an anticoagulant, it is transported to a laboratory or biorepository which either freezes the entire sample or aliquots thereof, or centrifuges the tube at room temperature to fractionate the sample into plasma, buffy coat and erythrocytes or red blood cells (RBCs). The most suitable blood fraction for DNA isolation is the buffy coat rich in leukocytes or white blood cells (WBCs), subdivided into

granulocytes, lymphocytes, and monocytes, as well as platelets or thrombocytes. However, in just 1 millilitre (mL) of human whole blood there are 39-57 million RBCs having no nucleus or DNA, with only 0.1% of blood cells, the nucleated WBCs, containing the analyte(s) of interest, DNA. Careful removal of the upper layer of plasma from a fractionated blood sample provides access to the buffy coat, often referred to as "crude" or "dirty" due to contamination of this WBC layer with RBCs and other blood components. From about 8 mL of blood in a standard 10 mL blood tube, 0.5-1.0 mL of buffy coat is withdrawn by hand or liquid-handling robotic systems. Subsequently, this crude buffy coat isolate is stored in a freezer or first treated with reagents that preferentially lyse RBCs, leaving WBCs intact.

[0005] In human blood, there exists an approximately one thousand-fold difference in the concentration of RBCs to WBCs. This presents not only an issue for the isolation of nucleic acid from vast numbers of collected fresh or frozen whole blood samples, the current storage technologies, particularly for large volumes of whole blood and blood derivates, are costly, inconvenient, impractical and inadequate. In general, there are four broad strategies for long- term nucleic acid preservation/storage: room temperature on a "dry" solid matrix, -20°C, - 80°C and -196°C (storage in liquid nitrogen). Sub-zero temperature storage requires costly equipment, such as cold rooms, freezers, electric generator back-ups, monitoring systems, and complex disaster recovery plans. Consequently, larger facilities are needed to house the necessary equipment and adequate technical staff must be employed to manage the specimens and associated equipment. Furthermore, it is common practice to store buffy coat and WBCs cryopreserved in liquid nitrogen which adds considerable cost. Power outages and remote areas without a reliable source of electricity and appropriate infrastructure are particularly problematic for the preservation of biospecimens. Without adequate generator back-up, power failures necessitate the relocation of frozen specimens and often lead to the loss of valuable samples.

[0006] Two of the common storage methods, dried and stored at room temperature and ultra- low temperature storage at -196°C, share a common mechanism where nucleic acid is maintained in a glassy or vitreous state in which molecules lose the ability to diffuse, thereby preventing chemical and nuclease degradation. However, if any moisture is added to the "dry state" or the temperature is raised above the glass transition temperature of water, movement and reactivity is re-established and damage to nucleic acid can occur.

[0007] Storage at -20°C to -80°C may provide adequate conditions for some applications depending on the quality and quantity of nucleic acid required and the time frame in which the sample needs to be stored. However, neither of these "freezer" conditions will maintain nucleic acid quality equivalent to maintenance at liquid nitrogen temperatures over extended periods of time. For long-term storage of blood, -80°C is recommended, since storage at - 20°C can result in lower yields and nucleic acid degradation.

[0008] In contrast to storage of specimens or DNA in solution at very low temperatures, dried storage is also possible. In addition to reducing molecular mobility, dehydration also removes water that can participate in hydrolytic reactions. Specimens and DNA can be dried, for instance, on FTA ^® Cards (Whatman ^® technology). Cells are lysed upon application to the treated card and nucleic acids are immobilized/stabilized for storage at room temperature, as long as moisture conditions are tightly controlled. Also, during dry storage, nucleic acid binds tightly to the fibers of the card, resulting in substantial losses of genetic material, particularly high molecular weight DNA, during the rehydration process. The additional handling required to recover nucleic acid from various solid supports can result in reduced nucleic acid yields, increased opportunity for the introduction of unwanted contaminants, and exposure to conditions that promote sample degradation; all of which are cost- and labour- intensive. Disaccharides, such as trehalose, have been employed for dry storage of samples to aid in the recovery of nucleic acid; however, such compounds serve as energy sources for undesirable microbial contaminants and subsequent degradation of desired nucleic acid. In addition, drying methods are not practical for the storage of large volumes of biological materials, such as whole blood and buffy coat.

[0009] Oftentimes, transportation of whole blood or whole blood derivatives is required. It is common practice for specialized carriers to transport such biological specimens in dedicated transport containers, providing a refrigerated environment using ice, dry ice or other freezing means, in order to maintain the cold-chain from collection to storage, and thus minimize or prevent WBC lysis and subsequent degradation of nucleic acid. However, adequate low temperatures often cannot be maintained for the extended periods of time required to transport samples, for instance, between countries or from remote locations to central processing facilities or biorepositories. Hence, the ability to transport and store biological specimens, particularly blood and buffy coat, at ambient and variable temperatures, while preserving nucleic acid in said specimens, would be highly advantageous.

[0010] Isolating nucleic acid from complex biological specimens, such as whole blood, is very labour intensive, repetitive, and consequently expensive. In addition, processing of biological specimens and successful isolation of nucleic acid requires the precision and attention to detail of a skilled technician. Biobanks or biorepositories often choose to store in freezers whole blood and or buffy coat, rather than store nucleic acid isolated from these specimens, in order to defer costs. Once an order is placed for nucleic acid from a particular donor(s), for example, frozen whole blood or buffy coat is thawed and nucleic acid isolated. However, ice crystals that formed during storage, followed by thawing of the specimen, produces significant cell damage and or lysis of cells. Damaged or lysed cells in the specimen are known to release nucleases which rapidly degrade both naturally-circulating, cell-free DNA (cfDNA) and nucleic acid released from damaged WBCs.

[0011] In addition, prior to the isolation of nucleic acid from whole blood and buffy coat fractions, the extra processing required to preferentially lyse RBCs and subsequent washing steps employed to remove lysed RBCs and various inhibitors found in blood samples, such as heme, iron, lactoferrin, immunoglobulin G (IgG), heparin and EDTA, result in the complete or near complete loss of both naturally-circulating, cfDNA and nucleic acid released from damaged WBCs. With repeated freezing/thawing of a specimen, genomic DNA is sheared and significant reductions in the yield and quality of nucleic acid are observed with each cycle. Remarkably, one cycle of freezing blood to -70°C and thawing to 37°C reduces DNA yield by more than 25% (Ross et al, 1990). In addition to damage by nucleases, aqueous solutions of DNA and biological samples are also susceptible to damage by oxidation, the rate of which is enhanced by the presence of trace metals (e.g. Fe ³⁺ , Cu ²⁺ ), found in blood, due to the production of free radicals via Fenton-type reactions. Hence, for the reasons outlined above, it would clearly be advantageous to store biological specimens, in particular the vast numbers of archived whole blood and buffy coat specimens, at ambient or room temperature for prolonged periods in a liquid composition which stabilizes all sources of nucleic acid and incorporates chelator agents to inhibit the action of deleterious nucleases and metal -catalyzed reactions.

[0012] The conventional methods of preserving nucleic acid in blood specimens during periods of transport and storage by the specimen at subzero temperatures is particularly problematic for non-invasive prenatal tests (NIPTs). Recent breakthroughs in technologies, for example, next-generation sequencing, and the discovery in the late 1990s that cell-free fetal DNA (cffDNA) comprises a generous 5-10% of the genetic material in a pregnant woman's bloodstream, has led to the introduction of NIPTs into clinical practice at an unprecedented pace. cffDNA is released into the pregnant woman's bloodstream when placental cells break down and can be readily isolated and screened from a simple blood draw done as early as 9- or 10-weeks gestation. These new NIPTs offer rapid turnaround of results, >99% specificity, and low (<0.1%) to nonexistent false-positive failure rates for the most common chromosomal defects, including Down (trisomy 21), Edwards (trisomy 18) and Patau (trisomy 13) syndromes, aneuploidies involving sex chromosomes which cause conditions that include Turner and Klinefelter syndromes and reveal the baby's sex. The present invention removes drawbacks of the known standard processes, ensuring that all nucleic acid present in a biospecimen, including cfDNA, cffDNA and cellular nucleic acid, is preserved in a time- and cost-effective manner at ambient temperature for extended periods and readily available for isolation and analysis. [0013] Some of the first DNA purification or extraction methods were based on the use of chaotropes which increase the solubility of molecules ("sal ting-in") by changing the structure of water and ultimately lyse cells and denature proteins. Recently, GW Fischer and LT Daum (Longhorn Vaccines & Diagnostics LLC) demonstrated that nucleic acid of a biological sample can be isolated and stabilized at ambient or elevated temperature when contacted with an aqueous mixture containing one or more chaotropes, detergents, reducing agents, chelating agents, and buffers (US Patent No. 8,084,443). However, chaotropic salts, such as guanidine thiocyanate, guanidine isocyanate, and guanidine hydrochloride are caustic and toxic and can lead to a reduction in quality and quantity of high molecular weight genomic DNA. Finally, prolonged exposure to such strong chaotropes might induce spurious oxidation of DNA, particularly at elevated temperatures during sample collection and transport. In addition, other methods for isolating and storing nucleic acids are known (see US Patent No. 8,470,536).

[0014] Thus, there exists a need for a method for the isolation of nucleic acid from biological samples that does not employ harmful, toxic reagents, and that employs fewer steps than conventional methods. In particular, there is a need to provide a method of isolating and storing nucleic acid from blood fractions, particularly the buffy coat, which improves on existing methods.

[0015] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY

[0016] An object of the present invention is to provide a method and aqueous composition for nucleic acid storage from blood fractions.

[0017] In accordance with one aspect of the present invention, there is provided a method for stabilizing nucleic acid in whole blood or a fraction thereof at ambient temperature. In one embodiment, the method comprises: collecting a whole blood sample from a donor; mixing the collected whole blood sample with an aqueous composition, preferably in the absence of a chaotrope, wherein the aqueous composition comprises a denaturing agent, chelating agent, and a buffering agent, wherein the pH of said aqueous composition is greater than 5.0, to form an aqueous mixture comprising the nucleic acid which is released from the whole blood sample; and storing said aqueous mixture at ambient temperature, wherein the nucleic acid is stabilized in the aqueous mixture.

[0018] In accordance with another aspect there is provided a method for stabilizing a nucleic acid in a total white blood cell isolate at ambient temperature which comprises: collecting a whole blood sample from a donor with an anti-coagulant; preferentially lysing red blood cells in the collected whole blood sample; centrifuging the red blood cell-lysed whole blood sample to form a red blood cell-depleted total white blood cell pellet; washing, at least once, total white blood cells in said red blood cell-depleted total white blood cell pellet; mixing the washed total white blood cell pellet with an aqueous composition preferably in the absence of a chaotrope, wherein the aqueous composition comprises a denaturing agent, chelating agent, and a buffering agent, wherein the pH of said aqueous composition is greater than 5.0, to form an aqueous mixture comprising the nucleic acid which is released from the total white blood cell pellet; and storing said aqueous mixture at ambient temperature, wherein the nucleic acid is stabilized in the aqueous mixture. [0019] In another aspect there is provided a method for stabilizing a nucleic acid in buffy coat at ambient temperature which comprises: collecting a whole blood sample from a donor with an anti-coagulant; centrifuging the whole blood sample to fractionate the collected sample into an upper layer (plasma), a middle layer (buffy coat), and a lower layer (red blood cells); removing and discarding the upper layer from said fractionated sample; isolating the middle or buffy coat layer from said fractionated sample; mixing said buffy coat isolate with an aqueous composition preferably in the absence of a chaotrope, wherein the aqueous composition comprises a denaturing agent, chelating agent, and a buffering agent, wherein the pH of said aqueous composition is greater than 5.0, to form an aqueous mixture comprising the nucleic acid which is released from the buffy coat isolate; and storing said aqueous mixture at ambient temperature, wherein the nucleic acid is stabilized in the aqueous mixture.

[0020] In yet another aspect there is provided a method for stabilizing a nucleic acid in a white blood cell isolate at ambient temperature which comprises: collecting a whole blood sample from a donor with an anti-coagulant; centrifuging the whole blood sample to fractionate the collected sample into an upper layer (plasma), a middle layer (buffy coat), and a lower layer (red blood cells); removing and discarding the upper layer from said fractionated sample; isolating the middle buffy coat layer from a fractionated whole blood sample; preferentially lysing red blood cells in the collected buffy coat isolate; centrifuging the red blood cell-lysed buffy coat isolate to form a red blood cell-depleted white blood cell pellet; washing, at least once, the white blood cells in said red blood cell-depleted white blood cell pellet; mixing the washed white blood cell pellet with an aqueous composition preferably in the absence of a chaotrope, wherein the aqueous composition comprises a denaturing agent, chelating agent, and a buffering agent, wherein the pH of said aqueous composition is greater than 5.0, to form an aqueous mixture comprising the nucleic acid which is released from the white blood cell pellet; and storing said aqueous mixture at ambient temperature, wherein the nucleic acid is stabilized in the aqueous mixture. [0021] In yet another aspect, there is provided a method of stabilizing viral RNA in a plasma sample at ambient temperature which comprises: collecting a whole blood sample from a donor with an anti-coagulant; centrifuging the whole blood sample to fractionate the collected sample into an upper layer (plasma), a middle layer (buffy coat), and a lower layer (red blood cells); isolating the upper plasma layer from the centrifuged whole blood sample; mixing the isolated upper plasma layer with an aqueous composition preferably in the absence of a chaotrope, wherein the aqueous composition comprises a denaturing agent, chelating agent, and a buffering agent, wherein the pH of said aqueous composition is greater than 5.0, to form an aqueous mixture comprising the nucleic acid which has been released from the isolated upper plasma layer; and storing said aqueous mixture at ambient temperature, wherein the nucleic acid is stabilized in the aqueous mixture.

[0022] The method of the present invention may further comprise: mixing the aqueous mixture with a composition for isolating the nucleic acid from the aqueous mixture and amplifying and quantifying the nucleic acid. In another embodiment, the method may further comprise mixing the aqueous mixture with a composition for isolating the RNA from the aqueous mixture; and amplifying and/or quantifying the RNA.

[0023] In certain embodiments, the blood fractions comprise, for example, white blood cells, buffy coat, or plasma.

[0024] For stabilizing nucleic acid in whole blood, an anti-coagulant may or may not be present. Typically, an anti-coagulant is used when stabilizing nucleic acid from blood isolates, such as white blood cell isolates, buffy coat, and plasma. [0025] In certain embodiments, the whole blood sample is collected in a collection vessel. The collection vessel can comprise a vial, a tube, a vacutainer, a specimen cup, a bottle, a blood tube, a bag, or variation thereof.

[0026] In certain embodiments, 9 volumes of the aqueous composition are mixed with 1 volume of the blood fraction, such as 9 volumes of aqueous composition to 1 volume of isolated blood layer. In certain embodiments, 1 volume of the aqueous composition is mixed with 1 volume of the blood fraction.

[0027] In certain embodiments, 9 volumes of the aqueous composition are mixed with 1 volume of whole blood. [0028] Typically, the aqueous composition comprises a denaturing agent, chelating agent, and a buffering agent. The pH of the aqueous composition is typically greater than 5.0.

[0029] In certain embodiments, the aqueous composition further comprises a salt.

Exemplary salts include lithium and sodium salts, such as sodium chloride, sodium acetate or lithium chloride, in an amount between about 0.001 and 1.0M, for example. [0030] In certain embodiments, the denaturing agent of the aqueous composition comprises at least one alcohol and/or one detergent. The alcohol can include one or more short-chain alkanols comprising methanol, ethanol, propanol, butanol, n-butanol, pentanol, hexanol, or any combination thereof which are present in an amount from about 0 to about 30%

(vol. /vol.). Exemplary detergents can include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, potassium dodecyl sulphate, sarkosyl, N-lauryl sarcosine, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, or any combination thereof, for example. Detergents can be present between 0 and about 10% (w/v), for example.

[0031] Ideally, the aqueous composition substantially maintains or stabilizes a nucleic acid for at least 12 weeks, or at least 30 weeks, at room temperature (i.e., from about 15°C to about 30°C). Ambient temperature can also refer to room temperature. The ambient temperature can also refer to the transport or storage temperature, such as for example between about -90°C to about 50°C. However, the aqueous composition can be used to store a nucleic acid sample from blood or a blood fraction at greater than room temperature, such as 50°C, for example. The aqueous composition can also be used to store a nucleic acid sample from blood or a blood fraction at a temperature less than room temperature, such as about -80°C or -196°C, for example. Other exemplary temperatures suitable for storage include about -196°C to about 50°C, or about -80°C to about 50°C.

[0032] Ideally, the aqueous mixture containing the nucleic acid and aqueous composition can be frozen and thawed, at least once, without negatively impacting DNA yield and quality.

[0033] In certain embodiments, the chelating agent of the aqueous composition comprises diethylenetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), trans-l^-diaminocyclohexane-N^N^N ¹ - tetraacetic acid (CDTA), l,2-bis(2-aminophenoxy)ethane-N,N,N ¹ ,N ¹ -tetraacetic acid (BAPTA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), N-(2- hydroxyethy ethylenediamine-N^^N^triacetic acid, or nitrilotriacetic acid (NT A). The chelating agent can be present, for example, in an amount of between about 1 mM and about 300 mM.

[0034] In certain embodiments, the buffering agent comprises Tris, sodium acetate, CDTA, glycine, borate, ADA, BES, PIPES, and HEPES. The amount of buffering agent in the aqueous composition can be between about 1 mM and about 1 M, for example.

[0035] In certain embodiments, the whole blood or blood fraction sample can comprise one or more animal cells, viruses, or bacteria that are substantially lysed upon contact with the aqueous composition. [0036] In accordance with another aspect, there is provided a whole blood and blood derivative storage system that comprises: a) a collection device or a collection vessel; and b) an amount of aqueous composition as described herein effective to substantially maintain or stabilize nucleic acid in whole blood and blood derivatives when mixed with said aqueous composition and stored at a temperature of from about -80°C to about 50°C for a period of at least 1 day, such as 2, 3, 4, 5, 6, 7, 14, 21, 28, 31, 270, 365 days, 3 months, 5 years or 25 years.

[0037] In certain embodiments, the system further comprises at least one storage vial.

[0038] In certain embodiments, the collection device comprises a swab, a capillary tube, a curette, a needle and syringe, a culture loop, a pipette, or a variation thereof; the collection vessel comprises a vial, a tube, a vacutainer, a specimen cup, a bottle, a blood tube, a bag, or variation thereof; and the storage vial comprises a cryovial, a tube, a cup, or variation thereof. The collection device or collection vessel can be adapted for collecting whole blood and fractionating whole blood into blood derivatives comprising plasma, buffy coat, white blood cells and red blood cell layers.

[0039] In particular, the present method differs from current practices to streamline workflow, minimize loss of nucleic acid from whole blood and blood derivatives caused by damage and/or degradation or from excessive handling, transport conditions and

contamination, facilitate fully automatic isolation of nucleic acid, store blood and blood derivatives at ambient temperature avoiding disadvantageous storage of large sample volumes in ultra-low temperature freezers, significantly impact sample storage

costs/overhead, and facilitate rapid nucleic acid isolation from stored samples.

[0040] The method and system described herein facilitate room temperature storage of DNA- containing components, eliminating the significant losses in DNA yield associated with the freezing/thawing of whole blood and blood derivates/fractions containing intact cells, and reduces the need to store samples at temperatures such as -80°C.

BRIEF DESCRIPTION OF THE FIGURES

[0041] For a better understanding of the present invention, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

[0042] Figure 1 shows an agarose gel of DNA from buffy coat samples stored for one month at room temperature or -80°C;

[0043] Figure 2 shows an agarose gel of DNA from white blood cell samples stored at room temperature, -20°C, -80°C or 50°C for 0, 3 and 6 weeks; [0044] Figure 3 shows an agarose gel of DNA from whole blood stored in the stabilization reagent of the present application for 5 years and for 10 months;

[0045] Figure 4 shows % DNA recovered over various freeze/thaw cycles; [0046] Figures 5a, 5b and 5c show agarose gels of DNA from buffy coat samples (SRA- protected and unprotected) of three donors over multiple (0-20x) freeze-thaw cycles;

[0047] Figure 6 shows an agarose gel of DNA from white blood cell fractions stored in the stabilizing reagent of the present application at room temperature for the indicated times; [0048] Figure 7 shows an agarose gel of DNA from buffy coat samples of 9 donors using a Promega® ReliaPrep gDNA MiniPrep System for DNA extraction;

[0049] Figure 8 shows an agarose gel of DNA from buffy coat samples of 9 donors using a QIAGEN® QIAamp DNA Blood Mini Kit;

[0050] Figure 9 shows an agarose gel of DNA from buffy coat samples of 9 donors using a Agencourt® GenFind v2 Blood & Serum gDNA Isolation Kit.

[0051] Figure 10 shows an agarose gel analysis of DNA purified from unprotected and protected buffy coat samples following 7 days in transit.

[0052] Figure 11 shows an agarose gel of purified DNA from buffy coat: cell lysis buffer samples stored at room temperature for 30 weeks. [0053] Figure 12 shows an agarose gel of purified DNA from buffy coat: cell lysis buffer samples stored at room temperature for 30 weeks, followed by 6 weeks at 50°C.

[0054] Figure 13 shows an agarose gel of purified DNA from buffy coat: cell lysis buffer samples stored at room temperature for 30 weeks, followed by 10 weeks at 50°C.

[0055] Figure 14 shows an agarose gel of DNA purified from whole blood stored for 1 day and 1 week under various conditions. Genomic DNA was isolated with Promega ReliaPrep Blood gDNA Miniprep System.

[0056] Figure 15 shows an agarose gel of DNA purified from whole blood stored for 4 weeks under various conditions. Genomic DNA was isolated with Promega ReliaPrep Blood gDNA Miniprep System or QIAamp DNA Blood Mini Kit. [0057] Figure 16 shows an agarose gel analysis of DNA purified from buffy coat fractions stored at room temperature in different volumes of stabilizing reagent. DETAILED DESCRIPTION

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0059] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0060] The term "comprising" as used herein will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and/or ingredient(s) as appropriate. [0061] The term "bodily fluid", as used herein, refers to a naturally occurring fluid from an animal, such as saliva, sputum, serum, plasma, blood and blood derivatives, cerebrospinal fluid, urine, mucus, gastric juices, pancreatic juices, feces, sweat, semen, products of lactation or menstruation, tears, or lymph.

[0062] The term "nucleic acid", as used herein, refers to a chain of nucleotides, including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically found in nature in chromosomes, chromatin, mitochondria, cytoplasm, ribosomes, bacteria, fungi and/or viruses.

[0063] The term "DNA polymerase", as used herein, refers to an enzyme that catalyzes a deoxyribonucleic acid synthesis via a primer binding and subsequent incorporation of nucleotides. Suitable polymerases include, but are not limited to, DNA polymerase I derived from E. coli, the Klenow fragment of DNA polymerase derived from E. coli, T4 DNA polymerase, Taq DNA polymerase, T. litoralis DNA polymerase, Τ ^Λ DNA polymerase and Pfu DNA polymerase.

[0064] The term "primer", as used herein, refers to an oligonucleotide acting as a starting point from which the synthesis begins in the presence of a DNA template, reagents for polymerization and so on. Although a primer is preferably single-stranded, double-stranded primers may also be used. When double-stranded primers are used, it is desirable to convert them into their single-stranded forms before use in an amplification reaction. A primer may be synthesized using well known methods, or may be isolated from an organism. [0065] The term "subject", as used herein, refers to an animal or human. Desirably, the subject is a mammal from which blood can be withdrawn for the purposes of nucleic acid isolation and detection. Most desirably, the subject is human.

[0066] As used herein, an "aqueous composition" is a composition for substantially stable storage of nucleic acid in a biological sample, in particular blood. The aqueous composition comprises a denaturing agent, chelating agent, and a buffering agent, wherein the pH of said composition is greater than 5.0. An "aqueous mixture" as used herein comprises the aqueous composition in admixture with a sample, such as a blood sample (or fraction(s) thereof), for example. [0067] In certain embodiments the chelating agent can be di ethyl enetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), trans- l^-diaminocyclohexane-NjNjN^N^tetraacetic acid (CDTA), l,2-bis(2- aminophenoxy)ethane-N,N,N ¹ ,N ¹ -tetraacetic acid (BAPTA), 1 ,4,7,10-tetraazacy clododecane- 1,4,7,10-tetraacetic acid (DOTA), N-(2-hydroxyethyl)ethylenediamine-N,N ¹ ,N ¹ -triacetic acid, or nitrilotriacetic acid (NT A).

[0068] In certain embodiments the denaturing agent can be at least one of an alcohol and a detergent. Exemplary alcohols include, for example, lower alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, or isobutanol, or the like.

[0069] In certain embodiments the denaturing agent can be a detergent or surfactant such as Triton X-100, sodium dodecyl sulphate (SDS), sodium laurilsulphate, sodium lauryl sulphate (SLS), sodium sarcosinate (sarkosyl), lithium dodecyl sulphate, sodium 1 -octane sul phonic acid, Tween 20, Tween 80, NP-40 and Briej 35, and the like.

[0070] In certain embodiments the buffering agent can be N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid (BES), 4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES), acetic acid or acetate (e.g. sodium acetate), citric acid or citrate, sodium

cyclohexane diaminetetraacetate (CDTA), malic acid, phthalic acid, succinic acid, histidine, pyrophosphoric acid, maleic acid, cacodylic acid, ββ'-Dimethylglutaric acid, carbonic acid or carbonate, 5(4)-Hydroxymethylimidazole, glycerol 2-phosphoric acid, ethylenediamine, imidazole, arsenic acid, phosphoric acid or phosphate, sodium acetate, 2:4:6-collidine, 5(4)- methylimidazole, N-ethylmorpholine, triethanolamine, diethylbarbituric acid,

tris(hydroxymethyl)aminomethane (Tris), 3-(N-Morpholino)propanesulfonic acid; 4- morpholinepropanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES), N-[tris(hydroxymethyl)methyl]-2- aminoethanesulfonic acid (TES), 4-(2-Hydroxyethyl)piperazine-l-propanesulfonic acid (EPPS), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), or combinations thereof. [0071] In certain embodiments the biological sample comprises whole blood and blood fractions, blood derivatives, red blood cell(s), white blood cell(s), white blood cell pellet, buffy coat, serum, plasma, red blood cell-depleted buffy coat, a granulocyte, a lymphocyte, a monocyte, a platelet/thrombocyte, lymph, cerebrospinal fluid, vaginal fluid, pleural fluid, ascites fluid, serosal fluid, pericardial fluid, amniotic fluid, umbilical cord blood/fluid, peritoneal fluid, perspiration/sweat, abdominal fluid, cell culture medium, a cell pellet, urine, a bacteria, a virus, a yeast cell, a fungus, a cell lysate, a homogenate or extract, a tissue lysate, a transformed cell line, an immortalized cell line, a biopsy specimen or a lysate, a DNA, and a RNA, or any combination thereof. The method of the invention is particularly suitable for the DNA-containing components, isolates or fractions in blood, buffy coats, leukocyte fractions, cell pellets and cell cultures, or any combination thereof.

[0072] The present application provides a method for substantially stabilizing one or a plurality of nucleic acid molecules that are present in a biological sample or bodily fluid, particularly a blood sample, comprising (a) admixing the biological sample with any of the above described compositions to obtain a mixture; and (b) maintaining the mixture without refrigeration and thereby substantially stabilizing said one or a plurality of nucleic acid molecules that are present in the biological sample, wherein degradation of the nucleic acid is substantially prevented.

[0073] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way. EXAMPLES

[0074] Materials & Methods

[0075] Preparation of whole blood and blood derivatives . DNA stabilization and isolation

[0076] In general, volunteers recruited for this study donated two blood tubes per donor. Approximately 7-8 mL of whole blood was collected from each donor into 10 mL EDTA-K Vacutainer tubes (#366643; 16 x 100 mm, 10.0 mL BD Vacutainer ^® plastic EDTA tube; Lavender BD Hemogard™ closure; Becton, Dickinson & Company). Blood tubes were inverted by hand five times and then placed on a rocker platform at room temperature to gently rock the tubes and ensure EDTA-K was fully dissolved. For studies requiring whole blood, 1 mL of blood was removed from one of each donor's tubes, mixed with 9 mL of the aqueous composition, alternatively referred to herein interchangeably as Stabilizing Reagent ("SR"), "SRA" or "HG", and stored at various temperatures.

[0077] Tubes containing whole blood were centrifuged at 1200xg for 10 minutes at room temperature to fractionate samples into plasma, buffy coat (5-1 Ox concentrated leukocytes) and packed red blood cell (RBC) or erythrocyte fractions. Plasma was gently removed from fractionated samples with a Pasteur pipette, leaving ~1 mL of plasma above the undisturbed buffy coat layer. For studies requiring the "crude" or "dirty" buffy coat fraction, a P200 micropipette (set at 100 μί) and "wide-bore" pipette tips were used to transfer approximately 0.5 mL of this fraction to a 15 mL conical tube. Buffy coat aliquots (0.5 mL) were frozen at - 20°C or prepared for room (15-30°C) and elevated temperature storage by mixing with approximately 4.5 mL or 9 volumes of Stabilizing Reagent (SR), e.g. Stabilizing Reagent A (SRA).

[0078] Leukocyte or white blood cell (WBC) pellets were prepared by removing red blood cells and other inhibitors from a) the isolated buffy coat fraction, b) whole blood, or c) the combined buffy coat and RBC fractions, with the addition of 3 volumes of RBC lysis solution (150 mM ammonium chloride (NH ₄ C1) in 10 mM sodium carbonate (NaHCOs)). Tubes were inverted ten times to initiate RBC lysis and incubated at room temperature for at least 15 minutes or until the solution cleared and turned dark red. To pellet intact white blood cells, tubes were centrifuged at 1500xg for 10 minutes at room temperature and the supernatant, containing haemoglobin and other inhibitors, was decanted. WBC pellets were washed at least once by resuspending the cells in 30 mL of 150 mM sodium chloride (NaCl, 0.9%). The suspension was split between three 15 mL conical tubes, centrifuged at 1500xg for 10 minutes at room temperature, and the supernatant was discarded. WBC pellets were gently resuspended in 2 mL of 150 mM NaCl, 500 aliquots of this suspension were transferred to four 1.5 mL Eppendorf tubes, centrifuged at 13,300rpm for 2 minutes at room temperature, and supernatant was aspirated from each tube. Washed WBC pellets were stored at -20°C until needed or mixed with 2 mL SR and then stored at subzero, room or elevated temperature. [0079] DNA purification from whole blood, freshly-prepared and frozen/archived buffy coat samples or white blood cell pellets in the present aqueous composition

[0080] Whole blood samples, freshly-prepared buffy coat (BC) or WBC samples in SRA (e.g., 1 volume sample: 9 volumes SRA), referred to herein as SRA-whole blood, SRA-BC and SRA- WBC samples, respectively, were stored at subzero, room or elevated temperature until required for DNA isolation using the Promega ReliaPrep™ Blood gDNA Miniprep System. Archived samples, maintained in storage at subzero temperatures (e.g. -20°C and - 80°C), were thawed for 2 minutes and mixed with 9 volumes of Stabilizing Reagent.

[0081] Using the Promega ReliaPrep™ Blood gDNA Miniprep System Technical Manual; follow the Instructions for Use of Products A5081, A5082; Literature # TM330, Revised 12/12 , with one exception; at "Step 3" of Promega ReliaPrep™ Blood gDNA Miniprep

System protocol, add 200 of SRA-whole blood, SRA-BC or SRA-WBC sample, instead of 200 of whole blood.

[0082] Determination of DNA concentration in purified samples [0083] Absorbance Determination of DNA Concentration [0084] DNA yields from purified whole blood, buffy coat and WBC samples, treated with or without stabilizing reagent under various conditions, were determined using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc.). A 2 μΐ _^ volume of each DNA sample was placed on the pedestal and scanned from 220 nm to 350 nm with absorbencies measured at 230 nm, 260 nm and 280 nm. Sample DNA concentration (ng^L), A ₂₆ o/A ₂₈ o ratio, and A ₂₆ o/A ₂₃₀ ratio were reported by the NanoDrop 2000c software. The total DNA yield per sample was calculated by multiplying the sample concentration by the respective DNA elution volume.

[0085] In general, the purity of nucleic acid samples is indicated by determining the ratio of spectrophotometric absorbance of the sample at 260nm to that of 280nm or A ₂₆ o/A ₂₈ o ratio. Nucleic acids and proteins have absorbance maxima at 260 and 280nm, respectively. Pure DNA preparations and DNA from biological samples with little protein contamination have A ₂ 6o A ₂₈ o and A ₂₆ o/A ₂₃₀ ratios around 1.8. Low A ₂₆ o/A ₂₈ o values may indicate protein contamination, while low A ₂₆ o/A ₂₃₀ ratios may indicate salt carryover or contamination with some solvents (e.g. phenol). Strong absorbance around 230nm can indicate that organic compounds or chaotropic salts are present in the purified DNA.

[0086] Fluorometric Determination of DNA Concentration

[0087] DNA yields from purified samples were quantified using the QuantiFluor® dsDNA System (Promega E2670) and the supplied Lambda dsDNA Standard (E259A; 100 μg/mL). The QuantiFluor® dsDNA System contains a fluorescent double-stranded DNA-binding dye (E258A; 504 nm Excitation/531 nm Emission) that enables sensitive quantitation of small amounts of double-stranded DNA (dsDNA). Triplicate 1 aliquots of each purified sample were processed according to the QuantiFluor® dsDNA System protocol, including a standard curve of the supplied Lambda dsDNA Standards [in triplicate; 0-50 ng/μυ] . Samples were processed in a black flat-bottomed 96 well microplate [655209; Greiner Bio-One] and fluorescence was measured using an Infinite M200 microplate reader [TEC AN].

[0088] DNA yields from purified samples were also quantified using PicoGreen ^® Fluorescent dye (Invitrogen, Cat.#P7581), a fluorescent double-stranded DNA-binding dye that enables sensitive quantitation of dsDNA in sub-nanogram amounts. Lambda DNA (Invitrogen, Cat.#25250-010) was used as a standard. Samples were processed in a black flat-bottomed 96 well microplate [655209; Greiner Bio-One] and fluorescence was measured using an Infinite M200 microplate reader [TEC AN].

[0089] Integrity of DNA in samples stored in stabilizing reagent

[0090] To assess DNA integrity, 50-100 ng from each purified SRA-whole blood, SRA-BC or SRA-WBC sample was separated on a 0.8% agarose gel by electrophoresis for 1 hour at 80 volts. The gel was stained in 1 μg/mL ethidium bromide in distilled water for 15 minutes at room temperature, rinsed and photographed on a UV transilluminator using a DigiDoc- IT™ imaging system (UVP LLC). The UltraRanger 1Kb DNA Ladder (300bp-24000bp; Norgen Biotek) was used as a size reference.

[0091] Amplification of DNA isolated from samples [0092] Purified DNA was evaluated in real-time or quantitative PCR (qPCR) for

amplification performance using primers targeting the single copy human thymidylate synthase gene (TYMS locus; NM001071.2). For each reaction, 50 ng of purified DNA was amplified in a 25 volume containing: lx PCR Buffer (20 mM Tris, 50 mM KC1), 2 mM MgCl ₂ , 200 μΜ dNTPs (Invitrogen), 50 μg/mL BSA (Sigma Aldrich), 1 μΜ SYT09 dye (Invitrogen), 0.4 μΜ each of Primer hTSml43F (5'-GCCCTCTGCCAGTTCTA-3') and hTSml43R (5 ' -TTCAGGCCCGTGATGT-3 ' ; Invitrogen), 1U Taq polymerase (Invitrogen). The amplification conditions for the TS143 target were: 1 cycle at 95 C for 5 minutes; 35 cycles at 95°C for 20 seconds, 55°C for 20 seconds, 72°C for 30 seconds; followed by 1 cycle at 72 C for 10 minutes. A melt curve program was included and consisted of: 1 cycle at 95 C for 30 seconds at a ramp rate of 4.4 C/second (no acquisition), 72 C for 10 minutes at a ramp rate of 2.2 C/second (no acquisition), 95 C at a ramp rate of 0.11 C/second (continuous acquisition). DNA samples were run in triplicate in a Corbett Rotorgene RG-6000 and C _t values for each sample calculated using the Rotorgene 6000 series software 1.7. The C _t value refers to the fractional cycle number at the point where the amplification curve crosses a threshold of detection. By setting a threshold line and calculating the intersection with each of the sample curves, the C _t values for each sample are established. The threshold line is set in the exponential phase of the run, significantly above the background level to avoid noise and below the onset of signal plateau in later cycles. Generally, the C _t value is inversely proportional to the amount of DNA in the sample. [0093] EXAMPLE 1 : Effect of different storage temperatures or laboratory workflows on DNA yield and integrity

[0094] The aqueous composition as described herein can be used in numerous workflows, e.g., starting with either freshly-prepared buffy coat or previously frozen, archived buffy coat samples. Different preparation and storage temperatures (room temperature, -80°C) for buffy coat samples mixed with the present aqueous composition do not significantly influence DNA recovery in terms of yield or concentration and quality. [0095] A method to store new in-coming buffy coat samples, as well as existing archived samples, at room temperature would be beneficial in reducing freezer costs, simplifying logistics, and providing additional sample protection for buffy coat. Stabilizing reagent (SR) of the present invention can be used to store buffy coat samples at room temperature, reducing risks associated with exposure to freeze-thaw. In cases where one prefers to freeze buffy coat samples, SR provides additional protection to samples by shielding them from degradation in cases of unexpected power failures. The present example looked at the yield and quality of DNA extracted from buffy coat samples in three different workflows:

• buffy coat sample preserved in SRA and stored for 1 month at room temperature (treatment A);

• buffy coat sample preserved in SRA and stored for 1 month at -80°C (treatment B);

• buffy coat sample frozen at -80°C for one month, and then thawed and mixed with SRA (treatment C) prior to analysis.

[0096] Specifically, three donors (S1-S3) were recruited for this study with 3 blood tubes drawn per donor. Approximately 7 mL of whole blood was collected from each donor per draw into 8 mL EDTA-K Vacutainer tubes (BD Vacutainer® plastic EDTA-K tubes;

Lavender BD Hemogard™ closure; Becton, Dickinson & Company). Samples were gently rocked at room temperature and centrifuged at 1200xg for 10 minutes at room temperature to fractionate samples into plasma, buffy coat, and RBC fractions. Plasma was gently removed from fractionated samples with a Pasteur pipette, leaving approximately 1 mL of plasma above the buffy coat layer. Using a P200 micropipette (set at 100 μί) and "wide-bore" pipette tips, 1 mL of each buffy coat fraction was transferred to a 15 mL conical tube. Hence, 3x15 mL conical tubes containing 1 mL buffy coat fractions were collected for each donor.

[0097] For each donor, A) 0.5 mL buffy coat sample was processed or lysed immediately by adding 4.5 mL of SRA and stored at room temperature for 4 weeks prior to analysis; B) 0.5 mL buffy coat sample was processed or lysed immediately by adding 4.5 mL of SRA, stored at -80°C for 4 weeks, thawed at room temperature prior to analysis; and C) 0.5 mL untreated buffy coat sample was stored at -80°C for 4 weeks, thawed for about 2 minutes at room temperature, lysed by adding 4.5 mL SRA prior to analysis. Triplicate aliquots (200 μί) of each SRA-BC sample per treatment (A-C) was then processed for DNA using the Promega ReliaPrep Blood gDNA Miniprep System according to the manufacturer's instructions and kit specific protocols (see Materials & Methods).

[0098] To assess DNA isolated from SRA-BC samples, DNA concentration and purity was estimated in triplicate using both absorbance and fluorometric methods (see Materials & Methods) for all 3 donors (see Table 1-1).

Table 1-1. Average DNA concentrations (ng^L) and 260nm/280nm absorbance ratios from 3 donors for DNA isolated from (A) fresh buffy coat (BC) samples stabilized in Storage Reagent A (SRA-BC) for 1 month at room temperature, (B) fresh buffy coat samples stabilized in SRA and stored for 1 month at -80 C, or (C)fresh buffy coat samples stored for 1 month at -80 C, thawed and then lysed in SRA at room temperature prior to analysis. Total DNA was isolated from SRA-BC samples with the Promega ReliaPrep Blood gDNA Miniprep System.

[0099] To assess DNA integrity, 100 ng from each purified SRA-BC sample was separated on a 0.8% agarose gel by electrophoresis and stained with ethidium bromide (Figure 1). High molecular weight genomic DNA bands (>23Kb) were visualized by UV illumination from samples from all three workflows (A-C) and from all three donors. The gel shows no DNA degradation of the samples from 3 donors (S I -S3) when comparing fresh SRA-BC samples stored at room temperature for 1 month (all "A" samples); SRA-BC samples stored at -80°C for 1 month (all "B" samples); and buffy coat samples stored at -80°C for 1 month, then thawed, mixed with SRA at room temperature prior to analysis (all "C" samples). In Table 1 - 2, purified DNA, evaluated in real-time or quantitative PCR (qPCR) using primers targeting the single copy human thymidylate synthase gene (TYMS locus; see Materials & Methods), showed equivalent amplification performance for all three workflows (A-C) and 3 donors.

Table 1-2. Averaged C _t values from TS 143 qPCR amplification curves for DNA isolated from (A) fresh buffy coat samples (3 donors) stabilized in SRA for 1 month at room temperature, (B) fresh buffy coat samples stabilized in SRA for 1 month at - 80 C or (C) fresh, untreated buffy coat samples stored for 1 month at -80 C and then stabilized in SRA at room temperature prior to analysis. Genomic DNA was isolated from the SRA-BC samples with the Promega ReliaPrep Blood gDNA Miniprep System.

[00100] EXAMPLE 2: Stability of DNA in white blood cells and whole blood samples exposed to a range of storage temperatures for extended periods of time.

[00101] Particularly during transport and storage of biological samples, such as whole blood and blood derivatives, maintenance of ideal temperature for sample and/or nucleic acid stability is problematic and costly. Presently, laboratories and biorepositories recommend storage of blood and blood fractions at -80°C to reduce the degree of cell lysis, cellular degradation, and DNA degradation which occurs at -20°C and higher temperatures. During transport of samples from the point of collection to the laboratory or biorepository, the cold chain must be maintained to preserve the integrity of the nucleic acids. The aqueous composition of the present invention alleviates these concerns and costly restrictions by enabling ambient temperature transport and storage of biological samples, such as whole blood and blood derivatives.

[00102] 2-1 White blood cells

[00103] Whole blood (~8 mL) was collected from 4 donors by venipuncture into two EDTA-K vacutainer tubes per donor. Tubes were inverted by hand five times and placed on a rocker platform until processed to keep cells in suspension, fully dissolve EDTA-K and prevent clumping. Vacutainer tubes were placed into a clinical centrifuge and spun at 1400xg for 15 minutes at room temperature to fractionate the blood into plasma, buffy coat and red blood cells. Plasma (approximately 4 mL) was removed from the upper fraction of each tube and discarded. About 1 mL of the buffy coat layer (plus a portion of the RBC fraction) from each tube was transferred into a 50 mL conical tube containing 4 mL of 0.9% [150mM] NaCl and three volumes (~15mL) of RBC lysis solution (150 mM NH ₄ C1 in 10 mM NaHCOs). Tubes were inverted gently 10 times and incubated at 37°C or room temperature until the solution colour changed from red (opaque) to dark red (clear red solution) due to RBC lysis and haemoglobin release. Tubes were centrifuged at 1400xg for 10 minutes at room temperature to pellet WBC (lymphocytes and monocytes). Supernatants were decanted and the cell pellets were gently resuspended in 20 mL 150 mM NaCl before centrifugation at 1400xg for 10 minutes at room temperature. The supernatant was decanted from each tube, RBC-free WBC pellets were gently resuspended in 4 mL 150 mM NaCl, and centrifuged to re-pellet WBC. In preparation for storage, washed WBC pellets were either A) mixed with SRA or B) stored as WBC pellets. [00104] Specifically, the WBC pellet in the first tube was mixed with 8 mL SRA, split into 2x4 mL aliquots in 15 mL conical tubes, and stored at room temperature and 50°C to demonstrate the stabilizing properties of the aqueous composition of the present invention. To replicate conventional storage methods, the washed WBC pellet in the second tube was resuspended in 150 mM NaCl to about 33 mL and then split into 32x1 mL aliquots in 1.5 mL microcentrifuge tubes. The microcentrifuge tubes were centrifuged at 4,000xg for 1 minute at room temperature, supernatants were removed and the cell pellets were stored either at -20°C or -80°C. Aliquots were "pulled" from the four different storage conditions for DNA analysis at 0, 3, 6, 12, 24, 36, 60, 120, 180, 240 and 360 week time points. [00105] At the indicated time points, WBC pellets stored at -20°C and -80°C, as well as 250 aliquots of SRA- WBC samples stored at room temperature and 50°C, were removed for each donor prior to analysis. WBC pellets were mixed with 75 150 mM NaCl and 150 SRA to lyse the cells. Proteinase K (-400 mg) was added to each aliquot, mixed and then incubated 1-2 hours in a 50 C water bath. After cooling aliquots at room temperature, 10 of prepIT™ ^» L2P (DNA Genotek, Ottawa, Canada) was added to each aliquot, mixed, incubated on ice for 10 minutes, and then centrifuged at 13,300rpm for 5 minutes at room temperature. The supernatant was transferred to a new 1.5 mL

microcentrifuge tube, 300 μΐ. of 95% ethanol was added to each tube and inverted several times to mix, before incubating at -20 C to precipitate DNA. To pellet the DNA, the tubes were centrifuged at 13,300rpm for 15 minutes at room temperature. Following removal of the supernatant, each pellet of DNA washed in 0.5 mL of 80% room temperature ethanol and centrifuged at 13,300rpm for 5 minutes at room temperature. Supernatant was carefully removed, the DNA pellet was air dried for 5-10 minutes at room temperature, and resuspended in 100 μΐ. TE. [00106] To visualize the integrity and quality of DNA isolated from Stabilizing Reagent-protected WBC samples and unprotected WBC pellets stored at the different temperatures, 100 ng of DNA from each purified sample was separated on a 0.8% agarose gel by electrophoresis (Figure 2). High molecular weight (>23Kb) DNA appears as a sharp band aligned with the upper most band of the 1Kb DNA ladder (300bp-24000bp). Figure 2 shows DNA isolated from a representative donor's (Donor 2) WBC samples stored at indicated temperatures (room temperature (RT), -20°C, -80°C or 50°C) for 0, 3 weeks and 6 weeks). WBC pellets were stored at -20°C and -80°C, while SRA- WBC samples were incubated at room temperature and 50°C for the indicated time periods. Three weeks storage at 50°C is equivalent to 12 weeks storage at RT; six weeks storage at 50°C is equivalent to 24 weeks storage at RT.

[00107] To assess further DNA isolated from SRA-WBC samples stored for prolonged periods at room temperature and 50°C, in contrast to unprotected WBC pellets stored at - 20°C and -80°C, DNA concentration and purity was estimated using both absorbance and fluorometric methods (see Materials and Methods) for all 4 donors and values were averaged (see Table 2-1).

Table 2-1. Average DNA concentrations (ng/μί), 260nm/280nm absorbance ratios and 260nm/230nm absorbance ratios from 4 donors for DNA isolated from SRA-

WBC samples stabilized at the indicated temperatures for the indicated time periods.

[00108] This example demonstrates that the aqueous composition of the invention stabilizes genomic DNA in WBC samples over an extremely broad range of "ambient" or elevated temperatures (room temperature to 50°C), commonly experienced during transport, handling and storage of biological samples, for long periods of time (several months). DNA isolated from SRA-WBC samples, even following long-term storage at extreme temperatures, remains high molecular weight (>23Kb), with no evidence of degradation by gel

electrophoresis (Figure 2). In addition, DNA purified from stored SRA-WBC samples was pure, as determined by absorbance, and its concentration remained the same at each storage temperature for at least 12 weeks. In stark contrast, DNA concentration of WBC pellets stored at conventional subzero temperatures (-20°C and -80°C) declined rapidly over a relatively short period of time. As noted in the prior art, -20°C storage of WBC pellets proved to be more detrimental than -80°C storage with respect to DNA concentration or yield.

[00109] 2-2 Whole blood

[00110] Whole blood was stored in SR at ambient temperature for a period of 5 years (Nov. 2008) and 10 months (Nov. 2012) prior to analysis of an aliquot. 500 aliquots from each of SRA-whole blood sample were processed using standard PCI

[phenol:chloroform:isoamyl alcohol] extraction and ethanol precipitation using the following procedure: a. 500 SRA-whole blood aliquot was treated with 20 Proteinase K (83.3 mg/mL) at 56°C for 10 minutes;

b. 500 PCI (phenol: chloroform: isoamyl alcohol (25:24: 1)) was added to each sample and vortexed vigorously for 30 seconds;

c. Samples were incubated at room temperature (RT) for 10 minutes and vortexed every 2minutes;

d. Centrifuged at 13,300rpm for 5 minutes at RT to separate aqueous and organic phases;

e. Carefully transfered the clear aqueous phase of each sample to a new 1.5 mL micro-centrifuge tube;

f. Added 1 μί glycogen (20 mg/ml) and 1/10 ^th volume 3M sodium

acetate pH5.5;

g. Added 2 volumes 95% ethanol and incubated overnight at -20°C; h. Centrifuged at 13,300rpm for 15 minutes at RT and carefully discarded the supernatant without disturbing or losing the nucleic acid pellet; i. Washed the pellet with 500 μί 70% ethanol and centrifuged at

13,300rpm for 5 minutes at RT;

j. Carefully discarded the supernatant without disturbing or losing the pellet;

k. Air dried the pellet in the open tube for 10 minutes at RT;

1. Resuspended the pellet in 50 μί lxTE and stored at 4°C or -20°C. [00111] Figure 3 shows an agarose gel of DNA purified from whole blood stored in SRA for prolonged periods of time. Genomic DNA was purified from whole blood samples which were stabilized in SRA at room temperature for 5 years (Nov 2008) and 10 months (Nov 2012), using standard Phenol:Chloroform:IsoAmyl Alcohol [25:24: 1] extraction and ethanol precipitation. Duplicate samples were analyzed by agarose gel electrophoresis as described in the Materials & Methods. High molecular weight genomic DNA bands (>23Kb) were visualized by UV illumination after staining the gel in ethidium bromide. Lanes 2 and 3 are samples stored at RT for 5 years. Lanes 4 and 5 are samples stored 10 months at RT.

[00112] EXAMPLE 3: Effect of stabilizing reagent on genomic DNA in buffy coat samples through multiple freeze-thaw cycles

[00113] Three donors were recruited for this study and 1 blood draw per donor was made. Approximately 7 mL of whole blood was collected from each donor per draw into a 10 mL EDTA-K Vacutainer tubes (#366643; 16 x 100 mm, 10.0 mL BD Vacutainer® plastic EDTA tube; Lavender BD Hemogard™ closure; Becton, Dickinson & Company). Samples were gently rocked at room temperature and centrifuged at 1200xg for 10 minutes at room temperature to fractionate samples into plasma, buffy coat, and RBC fractions. Plasma was gently removed from fractionated samples with a Pasteur pipette, leaving approximately 1 mL of plasma above the buffy coat layer. Using a P200 micropipette (set at 100 μί) and "wide-bore" pipette tips, a 0.5 mL buffy coat fraction was transferred to a 15 mL conical tube and diluted with an equal volume of [150 mM] NaCl or saline. A 0.5 mL aliquot of this buffy coat suspension was transferred to a 15 mL conical tube and prepared for storage by the addition of 4.5 mL SRA, vortexed and stored at -80 C after removal of "t=0 aliquot". To the remaining 0.5 mL buffy coat suspension, 4.5 mL 150 mM NaCl was added, vortexed and stored at -80 C. These samples were processed through 20 freeze/thaw cycles consisting of (i) freezing at -80 C for lhour, followed by (ii) thawing at 50 C for 15 minutes in a water bath. Aliquots (200 JL) of SRA-BC and buffy coat: 150 mM NaCl were collected before freezing at "t=0" and after freezing at 2, 4, 6, 8, 10, 15 and 20 cycles and then processed for DNA using the Promega ReliaPrep™ Blood gDNA Miniprep System according to the manufacturer's instructions and kit specific protocols. [00114] To assess DNA isolated from SRA-BC samples and buffy coat samples stored in saline at t=0 and following 2, 4, 6, 8, 10, 15 and 20 freeze-thaw cycles, DNA concentration and purity was estimated using both absorbance and fluorometric methods (see Materials & Methods) for all 3 donors.

[00115] Tables 3-1, 3-2 and 3-3 show average DNA concentrations (ng^L),

260nm/280nm absorbance ratios, and 260nm/230nm absorbance ratios from 3 donors for DNA isolated with the Promega ReliaPrep Blood gDNA Miniprep System from SRA- protected buffy coat samples and unprotected buffy coat samples after multiple freeze-thaw (F/T) cycles. "Avg. (no Ox)" represents the average ("Avg.") DNA concentration, A ₂₆ o, A ₂₈ o, ratio of A ₂₆ o/A ₂₈ o and ratio of A ₂₆ o/A ₂₃₀ for samples exposed to 2-20 F/T cycles, excluding baseline (t=0) samples not exposed to F/T conditions. Table 3-1. Effects of repeated freeze-thaw cycles on a buffy coat isolate prepared from whole blood (donor 1).

Table 3-2. Effects of repeated freeze-thaw cycles on a buffy coat isolate prepared from whole blood (donor 2).

150 inM

2 20 2.30 0.05 0.02 2.81 0.57 NaCl

Avg. 6.55 0.13 0.06 2.10 -0.81

Avg.

(no 2.67 0.05 0.02 2.12 -1.37

Ox)

Table 3-3. Effects of repeated freeze-thaw cycles on a buffy coat isolate prepared from whole blood (donor 3).

[00116] Figure 4 shows a comparison of the % DNA yield or recovered after each freeze-thaw cycle, compared to the original amount of DNA prior to freezing for SRA- protected buffy coat samples versus unprotected buffy coat samples. As shown in summary Table 3-4 and Figure 4, averaged SRA-BC samples only experienced a 1% loss of DNA after 2 freeze-thaw cycles, compared to a 75% loss of DNA in unprotected buffy coat samples after 2 freeze-thaw cycles as measured by fluorescence. The A ₂ 6o A ₂ 8o values for all samples are within expected range and greater than 1.9. The variability in the A ₂₆ o/A ₂₈ o values of the unprotected buffy coat samples was due to particulate cellular debris in these samples, whereas A ₂₆ o/A ₂₈ o values of SRA-BC samples are more consistent due to improved solubilization.

Table 3-4: The average DNA concentrations, yields and absorbance values from both the SRA-protected buffy coat samples (or SRA-BC) and the unprotected buffy coat samples (0.9%NaCl) after multiple freeze-thaw cycles (OX to 20X) were determined as described in the Materials & Methods.

[00117] To visualize the integrity and quality of DNA isolated from Stabilizing Reagent-protected and unprotected buffy coat samples after multiple freeze-thaw cycles, 100 ng of DNA from each purified sample was separated on a 0.8% agarose gel by

electrophoresis. High molecular weight (>23Kb) DNA appears as a sharp band aligned with the upper most band of the 1Kb DNA ladder (300bp-24000bp).

[00118] Figures 5a-c show agarose gel analyses of DNA isolated from 3 donors with the Promega ReliaPrep Blood gDNA Miniprep System from 200 aliquots of SRA- protected buffy coat samples and unprotected buffy coat (150 mM NaCl) samples after multiple (2x-20x) freeze-thaw (f/t) cycles. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol. Positive control ("Ox") samples were not exposed to freeze/thaw conditions prior to isolation of DNA; first lane of each agarose gel represents 1Kb DNA ladder (300bp-24000bp).

[00119] Figure 5a shows agarose gel analysis of DNA isolated from donor 1 buffy coat samples stored in the presence or absence ("unprotected") of Stabilizing Reagent ("SRA") and exposed to multiple freeze-thaw (f/t) cycles (Ox, 2x, 4x, 6x, 8x, lOx, 15x, and 20x). Figure 5b shows agarose gel analysis of DNA isolated from donor 2 buffy coat samples stored in the presence or absence ("unprotected") of Stabilizing Reagent ("SRA") and exposed to multiple freeze-thaw (f/t) cycles (Ox, 2x, 4x, 6x, 8x, lOx, 15x, and 20x). Figure 5c shows agarose gel analysis of DNA isolated from donor 3 buffy coat samples stored in the presence or absence ("unprotected") of Stabilizing Reagent ("SRA") and exposed to multiple freeze-thaw (f/t) cycles (Ox, 2x, 4x, 6x, 8x, lOx, 15x, and 20x). [00120] Purified DNA (50 ng) was evaluated in quantitative real-time PCR for amplification performance using primers targeting the single copy thymidylate synthase gene (TS143, TYMS locus; NM001071.2) as described in Materials & Methods. The qPCR results shown in Table 3-5 indicate that the genomic DNA in each SRA-BC sample is equivalent to a purified human genomic DNA reference/control sample (C _t values) even after the SRA-BC sample has been subject to 20 freeze-thaw cycles. The increase of C _t values for the unprotected buffy coat samples from 21.0 before freezing to 23.5 after 20 freeze-thaw cycles indicates decreasing amounts of nucleic acid for downstream analysis.

Table 3-5. After multiple freeze-thaw cycles, genomic DNA isolated from SRA- protected buffy coat samples (SRA-BC) and unprotected buffy coat samples (0.9% NaCl) were evaluated in qPCR for amplification performance using primers targeting the single copy tymidylate synthase gene (TS143) as described above.

[00121] This example demonstrates that DNA in buffy coat samples stored in SRA remains intact, high molecular weight and can be readily isolated with a commercial DNA extraction kit, even following 20 cycles of extreme temperature cycling between -80°C and 50°C. Strikingly, genomic DNA in buffy coat samples from the same donors and stored in saline was not stable through 20 freeze-thaw cycles. Following just 2 freeze-thaw cycles, a dramatic reduction in the intensity of the high molecular weight DNA band was observed for unprotected samples collected from all 3 donors. Measurement of DNA concentration and A ₂ 6o following freeze-thaw cycling confirmed that DNA in unprotected buffy coat samples was significantly degraded with each cycle, whereas DNA in SRA-BC samples was stable under the same extreme conditions. Specifically, only a 1% loss of DNA after 2 freeze-thaw cycles and a 9% loss of DNA after 20 freeze-thaw cycles for SRA-BC samples. In comparison, unprotected buffy coat samples lost 75% of DNA after 2 freeze-thaw cycles and a 91% loss after 20 freeze-thaw cycles. The aqueous composition of the present invention has great utility, since (i) buffy coat samples can be subjected to multiple freeze-thaw cycles due to repeated sampling, transport conditions, and unexpected power failures, and (ii) the capability to allow freeze-thaw cycles without DNA loss enables maximum flexibility and value from buffy coat samples for biorepositories and the like. The high yields of DNA isolated from SRA-BC samples were due to increased solubilization of DNA in these samples, leading to an increase in DNA available for recovery.

[00122] EXAMPLE 4: Stability of DNA in white cell pellets stored in stabilizing reagent at room temperature for 104 days. [00123] Using the same method outlined in example 2 above, WBC pellets were prepared from the whole blood of 5 donors. Each WBC pellet was resuspended in 3 mL 150 mM NaCl and then mixed with 3 mL SRA prior to storage at room temperature for at least 104 days.

[00124] At the indicated time points (0, 3, 7, 20, 30, 40, 84 and 104 days), a 500 aliquot of SRA-WBC sample from each donor was removed and DNA isolated. Proteinase K (-400 mg) was added to each aliquot, mixed and then incubated 1-2 hours in a 50 C water bath. After cooling aliquots at room temperature, \0 μΐ. of prepIT™ ^» L2P was added to each aliquot, mixed, incubated on ice for 10 minutes, and then centrifuged at 13,300rpm for 5 minutes at room temperature. The supernatant was transferred to a new 1.5 mL

microcentrifuge tube, 600 of 95% ethanol was added to each tube and inverted several times to mix, before incubating at -20 C to precipitate DNA. To pellet the DNA, the tubes were centrifuged at 13,300rpm for 15 minutes at room temperature. Following removal of the supernatant, each pellet of DNA washed in 0.5 mL of 80% room temperature ethanol and centrifuged at 13,300rpm for 5 minutes at room temperature. Supernatant was carefully removed, the DNA pellet was air dried for 5-10 minutes at room temperature, and resuspended in 100 TE.

[00125] To visualize the integrity and quality of DNA isolated from SRA-WBC samples stored at room temperature for prolonged periods of time, 50 ng of DNA from each purified 500 μΐ. SRA-WBC aliquot was separated on a 1% agarose gel by electrophoresis (Figure 6). Genomic DNA was purified from aliquots of a donor's SRA-WBC sample stored at room temperature for 104 days and analyzed at the indicated time points. High molecular weight (>23Kb) DNA appears as a sharp band aligned with the upper most band of the 1Kb DNA ladder. In this example, SRA demonstrates efficacy in stabilizing DNA in WBC pellets from whole blood stored at ambient temperature for at least 104 days. [00126] EXAMPLE 5: Plasma samples in stabilizing reagent

[00127] MicroRNAs (miRNAs), endogenous small non-coding RNAs that

posttranscriptionally regulate gene expression, are detected circulating in human plasma and serum. miRNAs, such as miR-223 and miR-21, are being increasingly used as novel biomarkers in the early-stage detection and diagnosis of several diseases, e.g. gastric cancer (GC), as conventional serum markers for GC, such as carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA), lack sufficient sensitivity and specificity to facilitate early detection (Li et al., 2012). Similarly, circulating RNA in plasma and serum can serve as both tumor- and fetal-specific markers for cancer detection and prenatal diagnosis, respectively (Tsui et al, 2006). Compared to the collection of tissue specimens for diagnostic purposes, plasma and serum are easy to access and noninvasive to obtain. This example investigates whether miRNA and mRNA can be detected and stabilized in SRA-plasma samples stored at room temperature. Healthy donors were recruited for this study; hence, nucleic acid levels in plasma samples are expected to be low and difficult to detect. [00128] In this example, whole blood was collected from 5 healthy donors by venipuncture into one EDTA-K Vacutainer (~8 mL per tube) for each donor. Tubes were inverted by hand x5 and placed on a rocker platform to keep cells in suspension and fully dissolve EDTA-K to prevent formation of micro-clumps. Whole blood in Vacutainers was placed into a clinical centrifuge and spun at 1500xg for 15 minutes at room temperature to separate blood into plasma, buffy coat and RBC fractions. Carefully, 4 mL of plasma was removed from the top fraction in each donor's tube and transferred to a 15 mL conical tube. To each tube containing 4 mL plasma, 4 mL SRA was added (SRA-plasma), tubes were inverted lOx to mix, and stored in a rack at RT prior to isolation of total nucleic acid. At indicated times, 500 uL aliquots were removed and processed for total nucleic acid. To isolate total nucleic acid from SRA-plasma samples, 500 aliquots were first incubated with Proteinase K (about 400 mg) overnight at 50°C in an air incubator. Next, samples were cooled to RT and processed to isolate (i) total RNA using TRIzol/TRIzol LS (Invitrogen) extraction and ethanol precipitation and (ii) DNA using standard PCI [25:24: 1] extraction and ethanol precipitation. [00129] Genomic DNA isolated from SRA-plasma samples stored for 0, 3, 5, 10, 15,

20 and 30 days were quantified using a PicoGreen ^® DNA quantitation method and evaluated in qPCR for amplification performance using primers targeting the single copy thymidylate synthase gene (TS143) as described in Materials & Methods. Table 5-1 demonstrates, as expected, that detectable amounts (>1 ng/μΐ.) of genomic DNA were not found in the SRA- plasma samples from the whole blood of healthy donors. However, qPCR results demonstrate that human DNA was detected in small amounts following amplification. Stable C _t values for TS143 in Table 5-1 indicate that DNA in SRA-plasma samples is stable for at least 30 days at room temperature.

Table 5-1: Total DNA yield and stabilization in SRA-plasma samples from 5 healthy donors.

¹ Total DNA yield from 500 SRA-plasma samples was below the level of detection for the PicoGreen DNA quantitation method used. C _t values suggest an average DNA concentration below 1 ng^L when compared to the C _t values for 50 ng human reference/control genomic DNA.

[00130] To synthesize cDNA from total RNA isolated from SRA-plasma samples, 11 (about 50 ng) of purified RNA was reverse transcribed using the Invitrogen Superscript III Reverse Transcriptase kit and protocol (Invitrogen 18080-044). To 11 of RNA sample, 1 μΐ, of random nonamers (NEB S1254S; 330 ng/μί) and 1 μί of RF lOmM dNTP mix (Invitrogen 10297-018) was added. All reactions were prepared on ice in a 200 μί PCR tube and then incubated in a thermal cycler. Samples were heated at 65°C for 5 minutes, followed by 4 minute incubation on ice. Master mix per sample (7 μί) used to synthesize cDNA consisted of 4 μΐ, of 5X First-Strand Buffer, Ι Ι, of 100 mM DTT, Ι Ι, of RNase Inhibitor (10 υ/μί) and ΙμΙ. of Superscript III (2000 υ/μί). 7 μί of master mix was added to each tube, mixed by pipetting, incubated for 5 minutes at 25°C, 50°C for 60 minutes, and the SSIII reaction was inactivated by heating at 70°C for 15min. cDNA was stored at -20°C.

[00131] cDNA was evaluated in quantitative PCR using Quantitect (mRNA) PCR primers (Qiagen) for beta-2-microglobulin (B2M) and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH). Master mix per reaction consisted of 2.5 of lOx PCR buffer, 1 μΐ, of 50 mM MgCl ₂ , 0.5 μΐ, of 10 mM dNTP, 2.5 μΐ, of 1 mg/mL BSA, 0.5 μΐ, Syto 9, 0.2 μί of Taq (5 υ/μί), and 12.8 μί nuclease-free water. In addition to 20 μί master mix, each reaction contained 3.0 μί of 10 μΜ F-primer, 3 μί of 10 μΜ R-primer and 2.0 μί of cDNA template. Two primer sets were evaluated with cDNA from SRA-plasma samples, beta-2- microglobulin or B2M (#QT00088935; 98bp fragment) and glyceraldehyde-3-phosphate dehydrogenase or GAPDH (#QT00079247; 95bp fragment). The reaction mixtures were incubated for lx [95°C for 5 minutes], 40x [95°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds], lx [72°C for 10 minutes]; melt analysis was lx [95°C for 10 seconds], lx [72°C for 1 minute], lx [95°C for 6 minutes]. Expression levels of microRNA-223 or miR- 223 in SRA-plasma samples were examined using two kits from Applied Biosystems or Invitrogen: TaqMan ^® MicroRNA Reverse Transcription Kit (Cat.#4366596) and TaqMan ^® microRNA Assay has-miR-223 (Cat.#4373075). The RNA and miRNA concentrations were calculated using standard curves constructed using synthetic RNA and miRNA.

[00132] Table 5-2 demonstrates that total RNA yield was below the level of detection; however, low levels of mRNA for two house-keeping genes (B2M and GAPDH) and miR- 223 were detected by quantitative reverse-transcriptase PCR (qRT-PCR) and stable at room temperature in SRA-plasma samples.

Table 5-2: Total RNA yield and detection of B2M mRNA, GAPDH mRNA, and miR223 in SRA-plasma samples from 5 healthy donors.

¹ Total RNA yield from 500 SRA-plasma samples was below the level of detection for the Ribogreen RNA quantitation method used. C _t values suggest an average RNA concentration below 1 ng^L when compared to the C _t values for known amounts of cDNA from 100 ng human lymphocyte total RNA. ² Mean C _t values for triplicate qRT-PCRs per cDNA/total RNA sample with the indicated primers.

[00133] EXAMPLE 6: DNA extracted from buffv coat stored in stabilizing reagent and purified with multiple third party extraction kits

[00134] Most bio-banks or bio-repositories have >10,000 legacy/archival/frozen buffy coat samples stored at -80 C and require/desire a stabilizing reagent and work-flow that permits or integrates with high-throughput automated DNA isolation systems Stabilizing Reagent, e.g. SRA, has been shown to be effective at stabilizing DNA in fresh buffy coat, frozen buffy coat and WBC pellets (data not shown here) prepared from either fresh or frozen buffy coat samples. In addition, SR has been shown to be compatible with a number of commercially available DNA isolation and purification systems, lending itself to high- throughput workflows.

[00135] Example 6A - Compatibility of the Promega ReliaPrep™ Blood gDNA MiniPrep System with fresh buffy coat samples in Stabilizing Reagent..

[00136] Sample collection, buffy coat preparation and DNA extraction

[00137] Nine donors were recruited for this study and two blood draws per donor were made. Approximately 7 mL of whole blood was collected from each donor into 2 x 10 mL EDTA-K Vacutainer tubes (#366643; 16 x 100 mm, 10.0 mL BD Vacutainer® plastic EDTA tube; Lavender BD Hemogard™ closure; Becton, Dickinson & Company). Samples were gently rocked at room temperature and then centrifuged at 1200 x g for 10 minutes at room temperature to fractionate samples into plasma, buffy coat and packed red blood cell fractions. Plasma was gently removed from fractionated samples with a Pasteur pipette, leaving ~1 mL of plasma above the buffy coat layer. Using a P200 micropipette (set at 100 μί) and "wide-bore" pipette tips, a 0.5 mL aliquot of the buffy coat fraction was transferred to a 15 mL conical tube and prepared for room temperature storage by the addition of 4.5 mL of stabilizing reagent A (SRA-BC). SRA-BC samples were stored at room temperature until required for gDNA isolation.

[00138] The Promega ReliaPrep™ Blood gDNA Miniprep system protocol was used for fresh buffy coat samples in stabilizing reagent A (SRA-BC). In the Promega ReliaPrep Blood gDNA Miniprep System Technical Manual; Instructions for use of Products A5081, A5082; Literature # TM330, Revised 12/12. At "Step 3" of Promega ReliaPrep Blood gDNA Miniprep system protocol, 200 of SRA-BC was added instead of 200 of blood.

[00139] Absorbance determination of DNA concentration

[00140] DNA yields from purified SRA-BC samples were determined using a

NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc.). A 2 μΐ _^ volume of each DNA sample was placed on the pedestal and scanned from 220 nm to 350 nm with absorbencies measured at 230 nm, 260 nm and 280 nm. Sample DNA concentration (ng^L), A260/A280 ratio, A260/A230 ratio were reported by the NanoDrop 2000c software. The total DNA yield per sample was calculated by multiplying the sample concentration by the respective DNA elution volume.

[00141] Fluorometric determination of DNA concentration [00142] DNA yields from purified SRA-BC samples were quantified using the

QuantiFluor® dsDNA System (Promega E2670) and the supplied Lambda dsDNA Standard (E259A; 100 μg/mL). The QuantiFluor® dsDNA System (a) contains a fluorescent double- stranded DNA-binding dye (E258A; 504 nm Excitation/531 nm Emission) that enables sensitive quantitation of small amounts of double-stranded DNA (dsDNA). Triplicate 1 μΐ aliquots of each purified SRA-BC sample were processed according to the QuantiFluor® dsDNA System protocol including a standard curve of the supplied Lambda dsDNA

Standards [in triplicate; 0-50 ng^L]. Samples were processed in a black flat-bottomed 96 well microplate [655209; Greiner Bio-One] and fluorescence measured using an Infinite M200 microplate reader [TEC AN].

[00143] Genomic DNA Integrity [00144] To assess DNA integrity, 100 ng from each purified SRA-BC sample was separated on a 0.8% agarose gel by electrophoresis for 1 hour at 80 volts. The gel was stained in 1 μg/mL ethidium bromide in distilled water for 15 minutes at room temperature, rinsed and photographed on a UV transilluminator using a DigiDoc-IT™ imaging system (UVP LLC). The UltraRanger 1Kb DNA Ladder (300bp-24000bp; Norgen Biotek) was used as a size reference for the genomic DNA samples.

[00145] DNA amplification

[00146] Purified DNA was evaluated in qPCR for amplification performance using primers targeting the single copy thymidylate synthase gene (TYMS locus; NM001071.2). For each reaction, 50 ng of purified genomic DNA was amplified in a 25 volume containing: lx PCR Buffer (20mM Tris, 50mM KC1), 2mM MgCl ₂ , 200 μΜ dNTPs (Invitrogen), 50 μg/mL BSA (Sigma Aldrich), 1 μΜ SYT09 dye (Invitrogen), 0.4 μΜ each of Primer hTSml43F and hTSml43R (Invitrogen), 1U Taq polymerase (Invitrogen). The amplification conditions for the TS143 target were: 1 cycle: 95°C for 5min; 35 cycles: 95°C for 20 seconds, 55°C for 20 seconds, 72°C for 30 seconds and 1 cycle 72°C for 10 minutes. A melt curve program was included and consisted of: 1 cycle 95°C for 30 seconds at a ramp rate of 4.4°C/second (no acquisition), 72°C for 10 minutes at a ramp rate of 2.2°C/second (no acquisition), 95°C at a ramp rate of 0.1 l°C/second (continuous acquisition). DNA samples were run in triplicate in a Corbett Rotorgene RG-6000 and C _t values for each sample calculated using the Rotorgene 6000 series software 1.7.

[00147] As shown in Table 6-1, DNA yields for SRA-BC samples from 9 donors differ, but are within the range expected for donor-to-donor variation. As expected, DNA yields by absorbance (NanoDrop) are higher than those observed by fluorescence

(QuantiFluor). Quantitation of DNA by absorbance is less time-consuming, but subject to interference by non-DNA moieties (proteins, RNA, carbohydrates, etc.). Quantitation of

DNA by fluorescence with DNA binding dyes (Quantifluor) is more accurate. The Nanodrop absorbance ratios shown in Table 6-1 indicate that the genomic DNA isolated from these SRA-BC samples is free of contaminants and inhibitors (organics, chaotropic salts, denaturants, etc. that absorb at 230 nm) that may inhibit or compromise molecular biology applications. The yield, concentration and A ₂₆ o/A ₂₈ o values are within the expected range for genomic DNA isolated with the Promega ReliaPrep gDNA MiniPrep System (Figure 1 and Figure 2 in the Promega ReliaPrep Blood gDNA Miniprep System Technical Manual; Instructions for use of Products A5081, A5082; Literature # TM330, Revised 12/12: pages 4- 5).

[00148] As shown in Figure 7, DNA was isolated from 200 aliquots of SRA-BC samples from 9 different donors using the Promega ReliaPrep™ gDNA MiniPrep System and analyzed by agarose gel electrophoresis. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol.

Table 6-1. DNA concentration, yield, and purity from SRA-BC samples processed using the Promega ReliaPrep gDNA MiniPrep System. Samples from 9 donors were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol.

[00149] Analysis of the genomic DNA isolated from the SRA-BC samples by agarose gel electrophoresis (Figure 8) shows high molecular weight genomic DNA in each sample and no evidence of degradation.

[00150] The quantitative real time PCR (qPCR) results shown in Table 6-2 indicate that the genomic DNA in each SRA-BC sample is equivalent to a purified human genomic DNA reference/control sample (C _t values). In addition, these results demonstrate that the genomic DNA in each SRA-BC sample is free of contaminants or inhibitors and is suitable for downstream molecular biology applications, including qPCR. Table 6-2: qPCR analysis of DNA from SRA-BC samples isolated using the Promega ReliaPrep gDNA MiniPrep System. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol. Aliquots (-50 ng) of the eluted DNA samples were analyzed by qPCR on a Corbett Rotorgene RG- 6000 as described in the Materials & Methods.

[00151] Example 6B - Compatibility of the OIAGEN QIAamp® DNA Blood Mini Kit with fresh buffy coat samples in Stabilizing Reagent.

[00152] Sample collection, buffy coat preparation and DNA extraction (see example 6A]

[00153] QIAGEN QIAamp DNA Blood Mini Kit protocol was used to isolate DNA from fresh buffy coat samples in stabilizing reagent. In the QIAamp DNA Mini and Blood Mini Handbook, Third Edition, June 2012; Protocol: DNA Purification from Blood or Body Fluids: Page 26, at "Step 2" of the QIAGEN QIAamp DNA Blood Mini Kit protocol (QIAamp DNA Mini and Blood Mini Handbook 06/2012, Protocol: DNA Purification from Blood or Body Fluids), 200 of SRA-BC was added, instead of 200 of blood.

[00154] As shown in Table 6-3, DNA yields for SRA-BC samples from 9 donors differ, but are within the range expected for donor-to-donor variation. The A ₂₆ o/A ₂₈ o values are higher than expected for genomic DNA alone. In this instance, the A ₂₆ o/A ₂₈ o values reflect the co-purification of DNA and RNA by the QIAamp Mini Spin Columns, since the optional RNAse A digestion step in the protocol was not performed for these samples. Table 6-3. DNA concentration, yield, and purity from SRA-BC samples isolated using the QIAGEN QIAamp DNA Blood Mini Kit. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol.

[00155] As shown in Figure 8, DNA was isolated from 200 μΐ, aliquots of SRA-BC samples from 9 different donors using the QIAGEN QIAamp™ DNA Blood Mini Kit and analyzed by agarose gel electrophoresis. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol. Analysis of the genomic DNA isolated from these SRA-BC samples shows high molecular weight genomic DNA in each sample and no evidence of degradation.

[00156] The quantitative real time PCR (qPCR) results shown in Table 6-4 indicate that the genomic DNA in each SRA-BC sample is equivalent to a purified human genomic DNA reference/control sample (C _t values). In addition, these results demonstrate that the genomic DNA in each SRA-BC sample is free of contaminants or inhibitors and is suitable for downstream molecular biology applications, including qPCR.

Table 6-4: qPCR analysis of DNA from SRA-BC samples isolated using the QIAGEN QIAamp DNA Blood Mini Kit. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol. Aliquots (-50 ng) of the eluted DNA samples were analyzed by qPCR on a Corbett Rotorgene RG-6000 as described in the Materials & Methods.

[00157] Example 6C - Compatibility of the Agencourt GenFind v2 Blood & Serum genomic DNA Isolation Kit with fresh buffy coat samples in Stabilizing Reagent.

[00158] Sample collection, buffy coat preparation and DNA extraction (same as

Example 6A)

[00159] Agencourt GenFind v2 Blood & Serum genomic DNA Isolation Kit protocol for fresh buffy coat samples in Stabilizing Reagent A was used. In the Agencourt GenFind v2 Blood & Serum genomic DNA Isolation Kit: Protocol 001072v001 ; Tube Purification

Procedure (For up to 400 of Blood/Serum): Page 8, at "Step 2" of the Agencourt GenFind v2 Blood & Serum genomic DNA Isolation Kit protocol: Tube Purification Procedure (For up to 400 μ∑ of Blood/Serum, added 400 of the fresh buffy coat samples in SRA, instead of 400 blood. [00160] As shown in Table 6-5, DNA yields for the individual SRA-BC samples from 9 donors differ, but are within the range expected for donor-to-donor variation. The Nanodrop absorbance (220 nm - 350 nm) scans (not shown) indicate that the genomic DNA isolated from the SRA-BC samples are free of contaminants and inhibitors (organics, chaotropic salts, denaturants, etc. that absorb at 230 nm) that may inhibit or compromise molecular biology applications. The A ₂₆ o/A ₂₈ o values are higher than expected for genomic DNA alone. In this instance, the A ₂₆ o/A ₂₈ o values likely reflect the co-purification of DNA and RNA by the Agencourt SPRI paramagnetic beads. The A ₂₆₀ /A ₂₃₀ values are lower than expected and are likely due to carry-over of some the Agencourt SPRI paramagnetic beads during the final elution step.

Table 6-5: DNA concentration, yield, and purity from SRA-BC samples isolated using the Agencourt GenFind v2 Blood & Serum genomic DNA isolation kit.

Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol.

[00161] As shown in Figure 9, DNA was isolated from 200 μΐ, aliquots of SRA-BC samples from 9 different donors using the Agencourt GenFind v2 Blood & Serum genomic DNA isolation kit and analyzed by agarose gel electrophoresis. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol.

Analysis of the genomic DNA isolated from the SRA-BC samples shows high molecular weight genomic DNA in each sample and no evidence of degradation.

[00162] The qPCR results shown in Table 6-6 indicate that the genomic DNA in each SRA-BC sample is equivalent to a purified human genomic DNA reference/control sample (C _t values). In addition, these results demonstrate that the genomic DNA in each SRA-BC sample is free of contaminants or inhibitors and is suitable for downstream molecular biology applications, including qPCR. Table 6-6: qPCR analysis of DNA from SRA-BC samples isolated using the

Agencourt GenFind v2 Blood & Serum genomic DNA isolation kit. Samples were processed with the supplied binding buffer, wash buffer and elution buffer according to kit protocol. Aliquots (-50 ng) of the eluted DNA samples were analyzed by qPCR on a Corbett Rotorgene RG-6000 as described in the Materials & Methods.

[00163] EXAMPLE 7: Shipping buffy coat samples at ambient temperature

[00164] Genetic research, genome-wide association studies and biobanks often require collection sites to ship patient samples to laboratories for analysis, to centralized locations for long-term storage, or to share samples between sites for collaborative studies. In cases where whole blood is being fractionated and the buffy coat is removed for future genetic analysis, the current standard for shipping buffy coat samples is to package and ship the sample on dry ice. This cold chain introduces specialized packaging which increases the size and weight of the package, requires additional labelling, and requires specialized couriers who are able to maintain the cold chain. All of these factors contribute to significant logistical challenges, increased packaging and shipping costs and the potential risk to valuable samples during transport.

[00165] A method of preserving the DNA in buffy coat at ambient temperature would be beneficial in reducing the costs and risks associated with transporting unpreserved samples collected for large population studies and long-term biobanking of samples. The stabilizing reagent of the present aqueous composition is a non-toxic reagent that is ideal for ambient temperature transport and room temperature archival storage of high molecular weight DNA in buffy coat samples. This example demonstrates the decrease in costs and time associated with shipping buffy coat in the present aqueous composition at ambient temperature versus shipping buffy coat samples on dry ice.

[00166] Packaging for frozen buffy coat sample [00167] A 1 mL frozen buffy coat sample in a 2 mL screw-cap O-ring tube was packaged in a biospecimen bag with an absorbent pad and surrounded with 12 kg of dry ice (sufficient quantity to maintain the cold chain for 5 days) and placed in a ULine S-7360 insulated foam shipping kit. This shipping kit includes a thick polystyrene foam container and an outer shipping carton. Two UN3373 biological substance, category B shipping labels and two transportation of dangerous goods dry ice labels marked with the weight of the dry ice were applied to the outside of the box to meet labelling and marking requirements for shipments that contain dry ice. Packaging material cost, including dry ice, was $106.37.

[00168] Packaging for buffy coat mixed with the present stabilizing reagent

[00169] A 0.5 mL buffy coat sample was mixed 1 : 1 with stabilizing reagent (SR) of the present invention in a 2 mL screw-cap O-ring tube. The tube was packaged in a ULine S- 18284 biological substance shipper. This shipper included a specimen transport bag, absorbent sheet, bubble wrap and the corrugated mailer pre-printed with the required labelling for UN3373 biological substances category B samples. Packaging materials cost $6.45. [00170] Shipping of packaged buffy coat samples

[00171] Each package was shipped from Ottawa, ON, Canada to a laboratory in Salt Lake City, UT, US using DHL Express service for next day delivery. Once the samples were received in Salt Lake City by the laboratory, they were then shipped back to Ottawa via FedEx International Priority service. Total shipping charges for buffy coat on dry ice cost $339.22, compared to $107.95 for shipping buffy coat in the present stabilizing reagent at ambient temperature. Although priority service was used, unexpected events occurred whereby the shipment was delayed at customs and the total transport time was 7 days; the buffy coat sample was exposed to a thawing cycle due to complete sublimation of the dry ice during transit. [00172] The unexpected, but common, delay in transit resulted in the degradation of the unprotected buffy coat sample due to the thawing and ambient temperature conditions of the sample, whereas the SR-protected buffy coat sample was not degraded when exposed to ambient temperature transport conditions (Figure 10). After 7 days in transit (7dit), equivalent volumes of genomic DNA preparations purified from the unprotected buffy coat sample and the protected buffy coat sample were loaded onto a 1 % agarose gel and run at 80 volts for 45 minutes at room temperature. High molecular weight genomic DNA bands (> 24 Kb; Figure 10) were visualized by UV illumination after staining the gel in ethidium bromide (1 μg/mL). [00173] This example demonstrates that the present stabilizing reagent significantly reduces packaging material and shipping costs for the transport of buffy coat samples. In addition, the buffy coat sample protected with the present reagent was not at risk of degradation due to delays in transport and consequential freeze-thaw exposure. The present stabilizing reagent protected high molecular weight genomic DNA in buffy coat during the highly variable and unpredictable conditions of international shipping.

[00174] EXAMPLE 8: Stability of buffy coat DNA in cell lysis buffers at ambient temperature.

[00175] Most genomic DNA isolation kits provide a cell lysis buffer as a primary reagent. Similar to the present invention, these buffers lyse cells, releasing the genomic DNA for subsequent isolation by various methodologies (e.g. alcohol precipitation, binding to columns or magnetic particles). Frequently, these commercial lysis buffers are composed of toxic chaotropic salts, such as guanidinium chloride, which among other functions, are known to inactivate degradative enzymes (e.g. deoxyribonuclease) and help to stabilize DNA. In the present example, lysis buffers from several different commercially-available DNA isolation kits were compared with the present Stabilizing Reagent (SR) for their ability to stabilize blood genomic DNA long-term at ambient temperatures.

[00176] Cell lysis buffers from the following DNA isolation kits were

evaluated: Gentra ^® PureGene ^® Blood Kit (QIAGEN, Cat. No. 158467); QIAamp ^® DNA Blood Mini Kit (QIAGEN, Cat. No. 51104); ReliaPrep™ Blood gDNA Miniprep System (Promega Corp., Cat. No. A5081); MagaZorb ^® DNA Mini -Prep Kit (Promega Corp., Cat. No. MB 1004); and Agencourt ^® GenFind™ v2 Blood & Serum Genomic DNA Isolation Kit (Beckman Coulter, Cat. No. #A41499).

[00177] Three donors (D1-D3) were recruited for this study with 2 blood tubes drawn per donor. Approximately 7 mL of whole blood was collected from each donor per draw into 8 mL EDTA-K Vacutainer tubes. Samples were centrifuged at 1200xg for 10 minutes at room temperature to fractionate samples into plasma, buffy coat and RBC fractions. Plasma was gently removed from the fractionated samples and 0.5 mL of each buffy coat fraction was transferred to a 15 mL conical tube. Two 0.5 mL buffy coat fractions from each donor were pooled and then resuspended in 6 mL of 0.9% NaCl. For each donor, this buffy coat suspension was split between 6x15 mL conical tubes (1 mL per tube). A different lysis buffer was added to each of the six conical tubes: 4 mL Stabilizing Reagent (SR); 4 mL Puregene (PG) Lysis Buffer; 2 mL GenFind (GF) v2 Lysis Buffer; 1 mL ReliaPrep (RP) Buffer CLD; 1 mL QIAamp (QIA) Buffer AL; and 1 mL MagaZorb (MZ) Lysis Buffer. The samples were mixed and stored long-term at room temperature (approximately 23°C). Following 30 weeks at room temperature, the samples were transferred to a 50°C oven for an additional 6-10 weeks to accelerate the aging process.

[00178] At the indicated times after storage, a 400 aliquot from each of the buffy coat: cell lysis buffer samples was incubated briefly with Proteinase K and then DNA was extracted using standard PCI [phenol: chloroform: isoamyl alcohol (25:24: 1)] extraction and ethanol precipitation. As outlined in Materials & Methods, purified DNA was evaluated using absorbance and fiuorometric methods (Tables 8-1, 8-2 and 8-3), DNA integrity was assessed using agarose gel analysis (Figures 11-13), and DNA was amplified in qPCR using primers for the single copy human TYMS gene (Tables 8-1, 8-2 and 8-3).

Table 8-1. Quantitation and amplification of DNA purified from buffy coat: cell lysis buffer samples of donor 1.

Table 8-2. Quantitation and amplification of DNA purified from buffy coat: cell lysis buffer samples of donor 2.

Donor Cell DNA Concentration DNA TS143 qPCR Average

Lysis (ng/μΐ.) by Concentration C _t value (n=3)

Buffer Picoquant (ng/μΐ.) by

Nanodrop

Day 1 at room temperature

D2 SR 51.1 63.6 20.65

D2 PG 43.6 65.2 21.07

D2 GF 35.9 42.7 19.51

D2 RP 73.3 75.6 20.39

D2 QIA 71.1 71.1 20.22

D2 MZ 67 79.4 19.90 hgDNA 50 19.05

30 weeks room temperature

D2 SR 58.7 73.4 26.22

D2 PG 64.2 101 no amp

D2 GF 52.5 52.8 25.26

D2 RP 74.9 85.5 25.59

D2 QIA 58.3 101.9 26.19

D2 MZ 29.6 13.3 28.85 hgDNA 50 24.25

30 weeks room temperature, 6 weeks 50°C

D2 SR 61.3 73.8 23.89

D2 PG 10 43.1 no amp

D2 GF 41 60.2 23.37

D2 RP 54.1 65.3 23.41

D2 QIA 54.4 99.7 23.59

D2 MZ 44.2 56.9 23.61 hgDNA 50 22.83

30 weeks room temperature, 10 weeks 50°C

D2 SR 65.3 73.5 21.46

D2 PG 6.3 20.8 no amp

D2 GF 42 56.8 20.7

D2 RP 53 64.2 20.81

D2 QIA 103.4 166.3 20.98

D2 MZ 44.3 60.3 21.1 hgDNA 50 21.15 Table 8-3. Quantitation and amplification of DNA purified from buffy coat: cell lysis buffer samples of donor 3.

[00179] Stabilizing reagent (SR) of the present invention stabilized high molecular weight genomic DNA in buffy coat samples stored at room temperature for 30 weeks (Figure 11) and 50°C for an additional 6 (Figure 12) and 10 weeks (Figure 13). Following 30 weeks storage at room temperature, agarose gel analysis (Figure 11) suggested that each commercial cell lysis buffer tested successfully stabilized genomic DNA in buffy coat samples, similar to the present aqueous composition. However, an additional stress of 50°C for 6 (Figure 12) and 10 (Figure 13) weeks resulted in significant fragmentation and degradation of high molecular weight DNA in buffy coat samples stored in cell lysis buffers from the Gentra Puregene Blood Kit, QIAamp DNA Blood Mini Kit and Agencourt GenFind v2 Blood & Serum Genomic DNA Isolation Kit. In contrast, high molecular weight DNA from the buffy coat of 3 donors remained intact in cell lysis buffers from ReliaPrep Blood gDNA Miniprep System and MagaZorb DNA Mini-Prep Kit with exposure to room temperature for 30 weeks and 50°C for 6 (Figure 12) and 10 weeks (Figure 13). Importantly, cell lysis buffers in both ReliaPrep and MagaZorb kits are reported (according to MSDS) to contain guanidinium chloride (25-50%), a toxic, strong chaotrope and protein denaturant.

[00180] Real-time PCR (Table 8-1 to 8-3) showed similar C _t values between 50 ng pure human genomic DNA and DNA isolated from buffy coatlysis buffer combinations, except for buffy coat in Puregene lysis buffer, indicating the isolation and amplification of pure DNA. DNA isolated from buffy coat sample treated with cell lysis buffer from Gentra Puregene Blood Kit did not amplify in real-time PCR suggesting the carryover of an inhibitor(s), in addition to DNA degradation (observed on agarose gel).

[00181] EXAMPLE 9: High molecular weight DNA in whole blood is stable in the present aqueous composition at ambient temperature.

[00182] Whole blood provides one of the most common sources of both high quality DNA and high quantity DNA for research and diagnostic purposes. Typically, blood is stored short-term at 4°C, ranging from a few days to a few weeks, whereas blood stored at room temperature needs to be processed right away. Long-term storage; however, usually involves blood being frozen, with a resultant loss in DNA yield. Many laboratories receive whole blood by mail and/or store blood samples prior to DNA extraction. Therefore, the effects of storage time (number of days from blood collection to DNA extraction) and temperature on DNA yield and quality are important. Remarkably, the present aqueous composition allows ambient temperature transport and storage of whole blood, without negative impact on DNA yield and quality. [00183] Whole blood storage

[00184] Two EDTA-K Vacutainer tubes of whole blood (~7ml/tube) were collected by venipuncture from each of 3 donors. Tubes were inverted by hand 5 times and placed on a rocker platform for 30 minutes to keep cells in suspension, fully dissolve EDTA-K and prevent clumping. For each donor, collected blood was split evenly between three 15 mL conical tubes. One conical tube was stored at room temperature (about 23°C), the second conical tube was stored at 4°C, and blood in the third conical tube was mixed 1 : 1 with an exemplary "HG" stabilizing solution [0.5 M sodium acetate, 0.2 M Tris, 10 mM CDTA, 1.0% SDS, 25 mM NaOH, 23.5 % ethanol, pH 9.6] of the present invention and then stored at room temperature up to one month. Following, 1 day, 1 week, 2 weeks, 3 weeks and 4 weeks storage, aliquots were removed from each conical tube for DNA extraction using the Promega ReliaPrep Blood gDNA Miniprep System and Qiagen QIAamp DNA Blood Mini Kit.

[00185] To visualize the integrity and quality of DNA isolated from whole blood stored under different conditions, 100 ng of DNA from each purified sample was separated on a 0.8% agarose gel by electrophoresis (Figures 14 and 15). DNA concentration and purity was estimated using both absorbance and fluorometric methods (see Materials & Methods) for all 3 donors (Table 9-1).

[00186] Purified DNA was evaluated by qPCR for amplification performance using primers targeting Homo sapiens TP53 gene exons 4-9 (2960 bp PCR product). For each reaction, 20 ng of purified DNA was amplified in a 20 volume containing: lx PCR Buffer (20 mM Tris, 50 mM KC1), 2 mM MgCl ₂ , 250 μΜ dNTPs (Invitrogen), 1 μΜ Syto 9 dye (Invitrogen), 5% DMSO, 10 μΜ (10 ριηοΐ/μΐ.) each of Primer 4FP53 (5'- CCTGAAAACAACGTTCTGGTAA-3') and 9RP53 (5'- TAGACTGGAAACTTTCCACTTG-3 ' ; Invitrogen), and 1 U Taq polymerase (Invitrogen). The amplification conditions for this target were: 1 cycle at 94°C for 5 minutes; 45 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds; followed by 1 cycle at 72°C for 10 minutes. Table 9-1. Averaged DNA concentrations (ng^L), 260nm/280nm absorbance ratios and 260nm/230nm absorbance ratios for DNA isolated following three storage conditions using two commercial extraction kits (Promega ReliaPrep Blood gDNA Miniprep System or QIAamp DNA Blood Mini Kit).

1 day 1 week 4 weeks

Pico- Pico- Pico-

Nanodrop Nanodrop Nanodrop quant quant quant

Samples

ng/ 260/ 260/ ng/μ ng/ 260/ 260/ ng/ 260/ 260/ ng/μί ng/μΐ.

280 230 L 280 230 280 230

Donor 1- blood

33.7 48.4 1.81 1.29 17.7 29.5 1.90 1.73 22.9 22.6 1.80 1.55

RT-

QIAamp

Donor 1- blood

36.8 61.0 1.74 0.82 23.5 37.1 1.94 1.66 39.0 40.2 1.87 1.85 4°C- QIAamp

Donor 1- blood

18.8 23.0 1.83 1.16 16.5 21.3 1.92 1.41 29.6 24.8 1.87 1.81 HG RT- QIAamp

Donor 2- blood

27.3 46.2 1.65 0.59 13.3 23.8 1.92 1.41 13.2 13.1 1.97 1.84

RT-

QIAamp

Donor 2- blood

32.0 45.2 1.80 1.15 20.7 35.7 1.88 1.16 35.1 35.7 1.82 1.82 4°C- QIAamp

Donor 2- blood

18.8 24.7 1.73 0.70 18.2 20.7 1.99 1.55 25.6 23.1 1.81 1.43 HG RT- QIAamp

Donor 3- blood

26.6 39.5 1.77 0.92 15.3 28 1.91 1.39 18.4 18.5 1.84 1.93

RT-

QIAamp

Donor 3- blood

24.1 34.2 1.84 1.39 17.1 30.8 1.96 1.37 24.6 29.3 1.68 0.78 4°C- QIAamp

Donor 3- blood

15.4 20.5 1.80 0.98 15.1 17.4 1.99 1.38 18.2 22.7 1.80 1.06 HG RT- QIAamp

Donor 1- blood

RT- 47.9 74.1 1.99 2.23 37.7 57.7 1.95 2.21 36.9 49.1 1.92 2.51

ReliaPrep

Donor 1- blood

4°C- 44.9 73.5 1.99 2.29 45.8 68.1 1.97 2.25 38.9 63.8 1.94 2.59

ReliaPrep

Donor 1- blood HG

RT- 25.5 30.0 2.01 2.42 23.4 34.6 1.95 2.24 31.0 35.6 1.94 3.08

ReliaPrep Donor 2- blood

45.0 66.5 1.98 2.32 29.8 49 1.98 2.21 32.0 44.4 1.91 2.74 RT-

ReliaPrep

Donor 2- blood

39.2 62.7 2.01 2.23 34.6 57.8 1.99 2.21 35.7 51.9 1.93 2.53 4°C-

ReliaPrep

Donor 2- blood HG

26.6 30.0 2.01 2.37 27.2 31.8 1.97 2.27 26.0 33.1 1.90 2.81 RT-

ReliaPrep

Donor 3- blood

32.3 51.4 2.03 2.22 29.2 42.9 1.99 2.2 24.3 35.7 1.88 2.70 RT-

ReliaPrep

Donor 3- blood

31.2 50.9 2.04 2.21 27.0 51.6 2.00 2.17 28.3 45.9 1.94 2.62 4°C-

ReliaPrep

Donor 3- blood HG

20.5 25.0 1.98 2.14 1 1.5 13.2 2.15 1.89 19.3 23.0 1.88 3.17 RT-

ReliaPrep

Table 9-2: Averaged C _t values from 49P53 qPCR amplification curves for DNA isolated from i) whole blood stored at room temperature, ii) whole blood stored at 4°C, or iii) whole blood stored 1 : 1 in "HG" stabilizing solution at room temperature, for 1 day and 4 weeks prior to analysis. Genomic DNA was isolated with Promega

ReliaPrep Blood gDNA Miniprep System.

Name Time Type c, AC,

20ng human gDNA Positive Control 22.52

No Template Control NTC 37.57

Donor 1 -blood RT-ReliaPrep 1 Day Unknown 22.61

Donor 1 -blood RT-ReliaPrep 4 Weeks Unknown 23.76 1.15

Donor 1 -blood 4°C-ReliaPrep 1 Day Unknown 22.29

Donor 1 -blood 4°C-ReliaPrep 4 Weeks Unknown 23.81 1.52

Donor 1 -blood HG RT-

1 Day Unknown 21.99

ReliaPrep

Donor 1 -blood HG RT-

4 Weeks Unknown 22.90 0.91

ReliaPrep

Donor 2-blood RT-ReliaPrep 1 Day Unknown 22.60

Donor 2-blood RT-ReliaPrep 4 Weeks Unknown 24.35 1.75

Donor 2-blood 4°C-ReliaPrep 1 Day Unknown 22.65

Donor 2-blood 4°C-ReliaPrep 4 Weeks Unknown 24.32 1.67

Donor 2-blood HG RT-

1 Day Unknown 22.56

ReliaPrep

Donor 2-blood HG RT-

4 Weeks Unknown 22.23 -0.33

ReliaPrep

Donor 3 -blood RT-ReliaPrep 1 Day Unknown 23.51 Donor 3 -blood RT-ReliaPrep 4 Weeks Unknown 25.28 1.77

Donor 3-blood 4°C-ReliaPrep 1 Day Unknown 22.58

Donor 3-blood 4°C-ReliaPrep 4 Weeks Unknown 22.90 0.32

Donor 3-blood HG RT-

1 Day Unknown 22.33

ReliaPrep

Donor 3-blood HG RT-

4 Weeks Unknown 22.28 -0.05

ReliaPrep

[00187] Apoptotic DNA fragmentation (DNA laddering) was evident in whole blood stored at room temperature and 4°C for 1 week to 1 month, whereas the blood mixed 1 : 1 with "HG" stabilizing solution showed no signs of DNA degradation at room temperature at any time point tested (Figures 14 and 15). In addition, high molecular weight DNA band intensity was also significantly diminished for whole blood stored at room temperature and 4°C for 2-4 weeks, whereas blood mixed 1 : 1 with "HG" stabilizing solution showed no signs of high molecular weight DNA band intensity decrease at room temperature (Figure 15). Overall DNA yield and purity (Table 9-1) remained stable during 4 weeks of storage.

Changes in qPCR with time and temperature (Table 9-2) were subtle, compared to DNA degradation readily observed in agarose gels, suggesting fragments (> 3 Kb) of DNA were still available for PCR in whole blood stored at room temperature or 4°C, in the absence of stabilizing solution.

[00188] DNA fragmentation and degradation observed in blood stored at room temperature and 4°C was prevented by treating freshly collected whole blood with stabilizing solution of the present invention. Advantageously, blood treated with stabilizing solution can be stored at room temperature until processing, freeing valuable space in fridges and freezers.

[00189] EXAMPLE 10: Ratios of buffy coat sample to stabilizing reagent

[00190] For optimal utility in the field, a stabilizing reagent should be tolerant of a wide range of volumes of blood and blood derivatives. This example examines the ratio of buffy coat sample to the present stabilizing reagent and its impact on DNA yield, concentration, purity and stability.

[00191] One donor was recruited for this "ratio" study and 4 blood draws into 4-10 mL

EDTA-K Vacutainer tubes (Becton, Dickinson & Company) were performed. These whole blood samples were pooled and dispensed back into Vacutainers as 2x 4 mL whole blood, 2x 6 mL whole blood and 2x 8 mL whole blood. These samples were gently rocked at room temperature for 15 minutes and centrifuged at 1200g for 10 minutes at room temperature to fractionate samples. Plasma was gently removed from fractionated samples with a Pasteur pipette, leaving 1 mL of plasma above the buffy coat layer. Using a P200 micropipette and "wide-bore" pipette tips, a 0.5 mL buffy coat fraction was transferred to a 15 mL conical tube for each 4 mL, 6 mL and 8 mL fractionated whole blood sample. Buffy coat (be) fractions were mixed with stabilizing reagent ("hg") in the following ratios:

[00192] Tubes with 4 mL whole blood (2x 0.5 mL buffy coat):

[00193] 0.5 mL aliquot of buffy coat (be) suspension was transferred to a 15 mL conical tube and mixed with 4.5 mL stabilizing reagent (hg), vortexed and stored at room temperature; [00194] 0.5 mL aliquot of buffy coat (be) suspension was transferred to a 15 mL conical tube and mixed with 1.5 mL stabilizing reagent (hg), vortexed and stored at room temperature;

[00195] Tubes with 6 mL whole blood (2x 0.5 mL buffy coat):

[00196] 0.5 mL aliquot of buffy coat (be) suspension was transferred to a 15 mL conical tube and mixed with 4.5 mL stabilizing reagent (hg), vortexed and stored at room temperature;

[00197] 0.5 mL aliquot of buffy coat (be) suspension was transferred to a 15 mL conical tube and mixed with 2.5 mL stabilizing reagent (hg), vortexed and stored at room temperature; [00198] Tubes with 8 mL whole blood (2x 0.5 mL buffy coat):

[00199] 0.5 mL aliquot of buffy coat (be) suspension was transferred to a 15 mL conical tube and mixed with 4.5 mL stabilizing reagent (hg), vortexed and stored at room temperature; [00200] 0.5 mL aliquot of buffy coat (be) suspension was transferred to a 15 mL conical tube and mixed with 3.5 mL stabilizing reagent (hg), vortexed and stored at room temperature.

[00201] Following 1 and 30 days storage at room temperature, aliquots (200 μί) were processed for genomic DNA isolation using Promega ReliaPrep Blood gDNA Miniprep System according to the manufacturer's instructions and kit specific protocols.

[00202] To visualize the integrity and quality of DNA isolated from buffy coat stored under different conditions, 100 ng of DNA from each purified sample was separated on a 0.8% agarose gel by electrophoresis (Figure 16), which shows agarose gel analysis of DNA purified from buffy coat fractions stored at room temperature in different volumes of stabilizing reagent. DNA concentration was estimated using both absorbance and

fluorometric methods (see Materials & Methods; Table 10-1). Purified DNA (20 ng) was evaluated in qPCR for amplification performance (Table 10-1) using primers targeting hTS143 (see Materials & Methods).

Table 10-1 : Average DNA concentrations (ng^L) and average C _t values from TS143 qPCR amplification curves for DNA isolated from buffy coat stored in different volumes of stabilizing reagent at room temperature for 1 and 30 days prior to analysis.

Sample Blood Ratio of Time DNA DNA TS143

Number volume buffy coat (days) Concentration Concentration Average C _t

(mL) (be): (ng μΐ.) by (ng μΐ.) by value stabilizing Picoquant Nanodrop

solution (hg)

43 4 1 :3 1 106.2 91.2 21.9

49 4 1 :9 1 36.8 31.8 22.0

65 6 1 :5 1 98.0 87.0 21.9

69 6 1 :9 1 51.7 49.5 22.0

87 8 1 :7 ¹ 82.8 70.3 22.1 89 8 1 :9 1 88.0 76.0 22.0

43 4 1 :3 30 101.9 71.6 22.0

49 4 1 :9 30 35.6 27.6 22.1

65 6 1 :5 30 83.3 69.1 21.9

69 6 1 :9 30 59.3 47.7 22.0

87 8 1 :7 30 89.3 74.3 22.0

89 8 1 :9 30 69.7 63.2 21.9

Human genomic DNA control

23.0

20 ng

[00203] Genomic DNA in buffy coat prepared from different volumes (4, 6, and 8 mL) of whole blood and mixed with different ratios (1 :3, 1 :5, 1:7 and 1 :9) of stabilizing reagent showed no loss in concentration/yield (Table 10-1) or quality (Figure 16) after 30 days at room temperature. Intact high molecular weight DNA was observed for each ratio of buffy coat to stabilizing reagent tested (Figure 16). Hence, the stabilizing reagent is tolerant of different volumes of buffy coat, preserving genomic DNA long-term at room temperature. qPCR values (Table 10-1) for purified DNA (20 ng input) were equivalent for 'low' (1 :3) and 'high' (1 :9) ratio buffy coat: stabilizing reagent samples, indicating that no

impurities/inhibitors were carried over into PCR.

[00204] EXAMPLE 11 : Compatibility of viral RNA with storage in stabilizing reagent at room temperature

[00205] The determination of viral load (the estimation of the amount of virus present in a bodily fluid) using mRNA as a marker is crucial for the detection and diagnosis of blood- borne diseases such as human immunodeficiency virus, type 1 (HIV-1). This example investigates whether viral RNA can be stored and stabilized in SR stored at room

temperature. A defined amount of inactivated HIV control RNA (Zeptometrix Corporation) was stored in SR and recovered using NucliSENS ^® (Biomerieux) RNA extraction. The NucliSENS ^® HIV-1 QT is an in vitro nucleic acid amplification test for the quantification of HIV-1 RNA in human plasma. The test can quantify HIV-1 RNA over the range of 176 to 3.47xl0 ⁶ copies/mL (NucliSENS ^® QT HIV-1, Biomerieux, Inc, Product Insert). The HIV-1 QT nucleic acid test (NAT) is one of 14 FDA approved NATs used for detection of HIV-1 in humans. [00206] EXAMPLE 11 A: Compatibility of the NUCLISENS^ magnetic bead extraction with viral RNA stored in stabilizing reagent

[00207] Inactivated HIV samples and RNA extraction

[00208] NATtrol™ HIV- 1 Linearity Panel (NATHIV 1 -LIN, Zeptometrix Corporation) is formulated with purified, intact, non-infectious virus particles supplied in a purified protein matrix that mimics the composition of a true clinical specimen, in this case a plasma sample. To assess compatibility of the HIV RNA with SR, 200μί of NATtrol was mixed with either ribonucl ease-free water, or SR. The samples were vortexed to mix NATrol HIV-1 virus with reagents, followed by a 10 minute incubation on ice. All samples were centrifuged for 5 minutes at 13,300 rpm and the supernatant transferred to a new ribonuclease-free

microcentrifuge tube.

[00209] RNA was extracted from each sample using the NucliSENS ^® magnetic bead extraction as per the manufacturer's instructions. Briefly, RNA samples were added to 2 mL tubes containing NucliSENS ^® lysis buffer and vortexed to mix. 50 of NucliSENS ^® silica beads were then added to each sample tube, vortexed to mix and incubated at room temperature for 10 minutes. All samples were centrifuged for 2 minutes at 1500 x g, and the supernatant removed without disturbing the pellet. The bead pellet was then resuspended in 400 NucliSENS ^® Wash Buffer 1 and transferred to a ribonuclease-free tube. All samples were washed as per the manufacturer's instructions, and finally eluted in 100 μΐ. elution buffer (supplied by manufacturer) for 5 minutes at 60°C. The final eluate was transferred to a fresh ribonuclease-free tube and stored at -80°C until further use.

[00210] cDNA Synthesis and amplification

[00211] HIV RNA was detected for each sample, in triplicate using a one-step RT- PCR (reverse transcriptase-PCR) reaction using the HIV primer/probe set from oasig (PrimerDesign genesig Quantification of HIV-1 Standard kit) and the manufacturer's instructions. In this reaction, cDNA is first synthesized from the total RNA isolated using the NucliSENS ^® beads, followed by amplification of the cDNA using the QIAgen OneStep RT- PCR kit reagents and the HIV-1 primer/probe mix from the PrimerDesign genesig

Quantification of HIV-1 Standard kit. The reaction mixture was assembled according to the following table: Table 11-1 : PCR Master mix

[00212] 12 of reaction mixture was added to each PCR reaction tube, and 8.0 of either sample (triplicates for each donor and blood control) or HIV control was subsequently added to the same tube. The HIV controls consisted of a dilution series of in activated HIV cDNA: 200, 20, 2 copies per μL. The PCR reaction mixtures were incubated in a RotorGene 6000 (Qiagen ^® ) thermal cycler according to the manufacturer's instructions:

Table 11-2: Thermal cycling conditions

[00213] The expected recovery of viral cDNA is ~ 3,000 copies (±~ 1,000) per reaction. Table 11-3 demonstrates that viral particles stored in stabilizing reagent is compatible with NucliSENS ^® RNA extraction and the subsequent detection of the cDNA. Table 11 -3 : Total RNA recovered (copies per reaction) from 200 HIV mixed with SR at room temperature.

[00214] EXAMPLE 1 IB: Stability of viral RNA in stored in stabilizing reagent at room temperature for 7 days [00215] Using the same method outlined in example 11 -A, inactivated HIV was mixed with SR, and stored at room temperature for 7 days. RNA extraction followed by cDNA synthesis and amplification was carried out as described in Example 11 -A, above. Table 1 1 -4 demonstrates that viral RNA can be successfully recovered after storage in SR for several days. Table 11 -4: Total RNA recovered from 200 μΐ. HIV mixed with SR and held at room temperature for 7 days, as compared to freshly prepared HIV/SR mixtures.

[00216] This example demonstrates that SR is compatible with standard, approved methods of viral RNA detection. Specifically it demonstrates that RNA contained in viral particles can be extracted from plasma stored in SR. Furthermore this example demonstrates that viral RNA can be stabilized in SR for up to 7 days. [00217] EXAMPLE 12: DNA extracted and purified from buffy coat stored in stabilizing reagent and used for genotyping.

[00218] It is common to assess genetic variability as an aspect of genetic research and large population studies. The use of single nucleotide polymorphism (SNP) genotyping has become increasingly useful in the characterization and diagnosis of disease. This example evaluates the performance DNA extracted from buffy coat samples stored in SR in a standard genotyping array.

[00219] Using the same methods outlined in the Materials and Methods section (Preparation of whole blood and blood derivatives, DNA stabilization and isolation) buffy coat preparations were obtained from healthy donors. Buffy coat fractions (4x 0.5 mL) for each donor were pooled into lx 15 mL conical tubes (total buffy coat preparation per donor ~2 mL) and stored at -80°C until required for experimentation. 1 mL of each buffy coat fraction pool per donor was added to 9 mL SR and stored at room temperature (RT) until required for experimentation. Aliquots of the buffy coat-SR mixture, or the frozen controls, were transferred to fresh tubes and DNA was extracted using the QIAsymphony DSP extraction protocols (Qiagen ^® , Blood 1000 DSP; Blood 400 DSP; Buffy Coat 400 DSP), according to the manufacturer's instructions. DNA extracted using this method was hybridized to the Illumina Infinium HumanCore 250K genotyping array, according to the manufacturer's specifications. Table 12-1 summarizes the genotyping call rates and concordance for frozen, untreated buffy coat samples and SR-treated samples.

Table 12-1 : Genotyping call rates and concordance for DNA isolated from frozen, untreated buffy coat samples and DNA isolated from buffy coat samples stabilized in SR at room temperature.

[00220] The genotyping concordance for buffy coat stored in SR as compared to frozen, untreated buffy coat samples was >99.99%. This demonstrates that storing buffy coat samples at room temperature in SR does not interfere with DNA performance on a standard genotyping platform.

[00221] All publications, patents and patent applications mentioned in this

Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.

[00222] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.